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Sample records for huntingtin htt protein

  1. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

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    Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  2. pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking

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    Pardo Raúl

    2010-06-01

    Full Text Available Abstract Background Huntingtin (htt is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s of full-length htt and its dysfunction(s associated with the disease. Results We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (Adaptable, RNAi Insensitive &Synthetic. It includes synthetic cDNA coding for full-length human htt modified so that: 1 it is improved for codon usage, 2 it is insensitive to four different siRNAs allowing gene replacement studies, 3 it contains unique restriction sites (URSs dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4 it contains multiple cloning sites at the N and C-ter ends and 5 it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells. Conclusion We report the validation of a synthetic gene encoding full-length htt protein that will facilitate

  3. pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking.

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    Pardo, Raúl; Molina-Calavita, Maria; Poizat, Ghislaine; Keryer, Guy; Humbert, Sandrine; Saudou, Frédéric

    2010-06-01

    Huntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease. We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (Adaptable, RNAi Insensitive &Synthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells. We report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its structure/function. This may help

  4. Quantification assays for total and polyglutamine-expanded huntingtin proteins.

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    Douglas Macdonald

    Full Text Available The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD. Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

  5. Truncated N-terminal huntingtin fragment with expanded-polyglutamine (htt552-100Q)suppresses brain-derived neurotrophic factor transcription in astrocytes

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    Linhui Wang; Fang Lin; Jin Wang; Junchao Wu; Rong Han; Lujia Zhu; Guoxing Zhang; Marian DiFiglia; Zhenghong Qin

    2012-01-01

    Although huntingtin (htt) can be cleaved at many sites by caspases,calpains,and aspartyl proteases,amino acid (aa) 552 was defined as a preferred site for cleavage in human Huntington disease (HD) brains in vivo.To date,the normal function of wild-type N-terminal htt fragment 1-552 aa (htt552) and its pathological roles of mutant htt552 are still unknown.Although mutant htt (mhtt) is also expressed in astrocytes,whether and how mhtt contributes to the neurodegeneration through astrocytes in HD remains largely unknown.In this study,a glia HD model,using an adenoviral vector to express wild-type htt552 (htt552-18Q) and its mutation (htt552-100Q) in rat primary cortical astrocytes,was generated to investigate the influence of htt552 on the transcription of brainderived neurotrophic factor (BDNF). Results from enzyme linked immunosorbent assay showed that the level of BDNF in astrocyte-conditioned medium was decreased in the astrocytes expressing htt552-100Q.Quantitative real-time polymerase chain reaction demonstrated that htt552-100Q reduced the transcripts of the BDNF Ⅲ and Ⅳ, hence, repressed the transcription of BDNF.Furthermore,immunofluorescence showed that aggregates formed by htt552-100Q entrapped transcription factors cAMP-response element-binding protein and stimulatory protein 1,which might account for the reduction of BDNF transcription.These findings suggest that mhtt552 reduces BDNF transcription in astrocytes,which might contribute to the neuronal dysfunction in HD.

  6. scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

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    Lai, Aaron Y; Lan, Cynthia P; Hasan, Salwa; Brown, Mary E; McLaurin, Joanne

    2014-02-07

    Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-β peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

  7. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes

    Institute of Scientific and Technical Information of China (English)

    Linhui Wang; Fang Lin; Junchao Wu; Zhenghong Qin

    2009-01-01

    Huntington's disease (HD) is caused by an expansion of polyglutamine tract in N-terminus of huntingtin (htt).The mutation of htt leads to dysfunction and premature death of striatal and cortical neurons. However, the effects of htt mutation on glia remain largely unknown.This study aimed to establish a glia HD model using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 amino acids (htt552) in rat primary cortical astrocytes. We have eval-uated optimal conditions for the infection of astrocytes with adenovirai vectors, and the kinetics of the expression of htt552 in astrocytes. The majority of astroeytes expressed the transgene after infection. At 24 h post-infection, the highest rate of infection was 89 + 3% for the wild-type (htt552-18Q) with a multiplicity of infection (m.o.i.) of 80, and the highest rate of infection was 91 +4% for the mutant type (htt552-100Q) with the same viral dose. The duration of expression of htt552 lasted for about 7 days with a relatively high level from 1 to 4 days post-infection. Mutant huntingtin (htt552-100Q) pro-duced the characteristic HD pathology after 3 days by the appearance of cytoplasmic aggregates and intranue-lear inclusions. The result of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu mbromide)assay showed that the inhibition of viability by virus on astrocytes was also dose-dependent. To obtain high infection rate and low toxicity, the viral dose with an m.o.i, of 40 was optimal to our cell model. The present study demonstrates that adenovirai-mediated expression of mutant htt provides an advantageous system for his-tological and biochemical analysis of HD pathogenesis in primary cortical astrocyte cultures.

  8. Identification of binding sites in Huntingtin for the Huntingtin Interacting Proteins HIP14 and HIP14L.

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    Shaun S Sanders

    Full Text Available Huntington disease is an adult onset neurodegenerative disease characterized by motor, cognitive, and psychiatric dysfunction, caused by a CAG expansion in the HTT gene. Huntingtin Interacting Protein 14 (HIP14 and Huntingtin Interacting Protein 14-like (HIP14L are palmitoyl acyltransferases (PATs, enzymes that mediate the post-translational addition of long chain fatty acids to proteins in a process called palmitoylation. HIP14 and HIP14L interact with and palmitoylate HTT and are unique among PATs as they are the only two that have an ankyrin repeat domain, which mediates the interaction between HIP14 and HTT. These enzymes show reduced interaction with and palmitoylation of mutant HTT, leading to increased mutant HTT inclusion formation and toxicity. The interaction between HIP14 and HTT goes beyond that of only an enzyme-substrate interaction as HTT is essential for the full enzymatic activity of HIP14. It is important to further understand and characterize the interactions of HTT with HIP14 and HIP14L to guide future efforts to target and enhance this interaction and increase enzyme activity to remediate palmitoylation of HTT and their substrates, as well as to understand the relationship between the three proteins. HIP14 and HIP14L have been previously shown to interact with HTT amino acids 1-548. Here the interaction of HIP14 and HIP14L with N- and C-terminal HTT 1-548 deletion mutations was assessed. We show that HTT amino acids 1-548 were sufficient for full interaction of HTT with HIP14 and HIP14L, but partial interaction was also possible with HTT 1-427 and HTT 224-548. To further characterize the binding domain we assessed the interaction of HIP14-GFP and HIP14L-GFP with 15Q HTT 1-548Δ257-315. Both enzymes showed reduced but not abolished interaction with 15Q HTT 1-548Δ257-315. This suggests that two potential binding domains exist, one around residues 224 and the other around 427, for the PAT enzymes HIP14 and HIP14L.

  9. Investigating Mutations to Reduce Huntingtin Aggregation by Increasing Htt-N-Terminal Stability and Weakening Interactions with PolyQ Domain

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    Mazza-Anthony, Cody; Waldispühl, Jérôme

    2016-01-01

    Huntington's disease is a fatal autosomal genetic disorder characterized by an expanded glutamine-coding CAG repeat sequence in the huntingtin (Htt) exon 1 gene. The Htt protein associated with the disease misfolds into toxic oligomers and aggregate fibril structures. Competing models for the misfolding and aggregation phenomena have suggested the role of the Htt-N-terminal region and the CAG trinucleotide repeats (polyQ domain) in affecting aggregation propensities and misfolding. In particular, one model suggests a correlation between structural stability and the emergence of toxic oligomers, whereas a second model proposes that molecular interactions with the extended polyQ domain increase aggregation propensity. In this paper, we computationally explore the potential to reduce Htt aggregation by addressing the aggregation causes outlined in both models. We investigate the mutation landscape of the Htt-N-terminal region and explore amino acid residue mutations that affect its structural stability and hydrophobic interactions with the polyQ domain. Out of the millions of 3-point mutation combinations that we explored, the (L4K E12K K15E) was the most promising mutation combination that addressed aggregation causes in both models. The mutant structure exhibited extreme alpha-helical stability, low amyloidogenicity potential, a hydrophobic residue replacement, and removal of a solvent-inaccessible intermolecular side chain that assists oligomerization. PMID:28096892

  10. Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington's disease patients.

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    Wild, Edward J; Boggio, Roberto; Langbehn, Douglas; Robertson, Nicola; Haider, Salman; Miller, James R C; Zetterberg, Henrik; Leavitt, Blair R; Kuhn, Rainer; Tabrizi, Sarah J; Macdonald, Douglas; Weiss, Andreas

    2015-05-01

    Quantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant huntingtin protein (mHTT), the cause of Huntington's disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously. We developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts. This SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT. We have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD. Not applicable. CHDI Foundation Inc.; Medical Research Council (MRC) UK; National Institutes for Health Research (NIHR); Rosetrees Trust; Swedish Research Council; and Knut and Alice Wallenberg Foundation.

  11. Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein.

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    Melone, Mariarosa A B; Calarco, Anna; Petillo, Orsolina; Margarucci, Sabrina; Colucci-D'Amato, Luca; Galderisi, Umberto; Koverech, Guido; Peluso, Gianfranco

    2013-01-01

    Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild-type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

  12. Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell-type-specific

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    Jansen, Anne H P; van Hal, Maurik; Op den Kelder, Ilse C; Meier, Romy T; de Ruiter, Anna-Aster; Schut, Menno H; Smith, Donna L; Grit, Corien; Brouwer, Nieske; Kamphuis, Willem; Boddeke, H W G M; den Dunnen, Wilfred F A; van Roon, Willeke M C; Bates, Gillian P; Hol, Elly M; Reits, Eric A

    2017-01-01

    Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions cou

  13. Huntingtin interacting proteins are genetic modifiers of neurodegeneration.

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    Linda S Kaltenbach

    2007-05-01

    Full Text Available Huntington's disease (HD is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to

  14. SUMO-2 and PIAS1 modulate insoluble mutant huntingtin protein accumulation.

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    O'Rourke, Jacqueline Gire; Gareau, Jaclyn R; Ochaba, Joseph; Song, Wan; Raskó, Tamás; Reverter, David; Lee, John; Monteys, Alex Mas; Pallos, Judit; Mee, Lisa; Vashishtha, Malini; Apostol, Barbara L; Nicholson, Thomas Peter; Illes, Katalin; Zhu, Ya-Zhen; Dasso, Mary; Bates, Gillian P; Difiglia, Marian; Davidson, Beverly; Wanker, Erich E; Marsh, J Lawrence; Lima, Christopher D; Steffan, Joan S; Thompson, Leslie M

    2013-07-25

    A key feature in Huntington disease (HD) is the accumulation of mutant Huntingtin (HTT) protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

  15. SUMO-2 and PIAS1 Modulate Insoluble Mutant Huntingtin Protein Accumulation

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    Jacqueline Gire O’Rourke

    2013-07-01

    Full Text Available A key feature in Huntington disease (HD is the accumulation of mutant Huntingtin (HTT protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

  16. A huntingtin peptide inhibits polyQ-huntingtin associated defects.

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    Yoan Arribat

    Full Text Available BACKGROUND: Huntington's disease (HD is caused by the abnormal expansion of the polyglutamine tract in the human Huntingtin protein (polyQ-hHtt. Although this mutation behaves dominantly, huntingtin loss of function also contributes to HD pathogenesis. Indeed, wild-type Huntingtin plays a protective role with respect to polyQ-hHtt induced defects. METHODOLOGY/PRINCIPAL FINDINGS: The question that we addressed here is what part of the wild-type Huntingtin is responsible for these protective properties. We first screened peptides from the Huntingtin protein in HeLa cells and identified a 23 aa peptide (P42 that inhibits polyQ-hHtt aggregation. P42 is part of the endogenous Huntingtin protein and lies within a region rich in proteolytic sites that plays a critical role in the pathogenesis process. Using a Drosophila model of HD, we tested the protective properties of this peptide on aggregation, as well as on different polyQ-hHtt induced neuronal phenotypes: eye degeneration (an indicator of cell death, impairment of vesicular axonal trafficking, and physiological behaviors such as larval locomotion and adult survival. Together, our results demonstrate high protective properties for P42 in vivo, in whole animals. These data also demonstrate a specific role of P42 on Huntington's disease model, since it has no effect on other models of polyQ-induced diseases, such as spinocerebellar ataxias. CONCLUSIONS/SIGNIFICANCE: Altogether our data show that P42, a 23 aa-long hHtt peptide, plays a protective role with respect to polyQ-hHtt aggregation as well as cellular and behavioral dysfunctions induced by polyQ-hHtt in vivo. Our study also confirms the correlation between polyQ-hHtt aggregation and neuronal defects. Finally, these results strongly suggest a therapeutic potential for P42, specific of Huntington's disease.

  17. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis.

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    Culver, Brady P; Savas, Jeffrey N; Park, Sung K; Choi, Jeong H; Zheng, Shuqiu; Zeitlin, Scott O; Yates, John R; Tanese, Naoko

    2012-06-22

    Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.

  18. Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments.

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    David C Butler

    Full Text Available Huntington's disease (HD is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG(n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt, formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs, expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1 significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.

  19. Huntingtin interacting proteins 14 and 14-like are required for chorioallantoic fusion during early placental development.

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    Sanders, Shaun S; Hou, Juan; Sutton, Liza M; Garside, Victoria C; Mui, Katherine K N; Singaraja, Roshni R; Hayden, Michael R; Hoodless, Pamela A

    2015-01-15

    Huntington disease (HD) is an adult-onset neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms that is caused by a CAG expansion in the HTT gene. Palmitoylation is the addition of saturated fatty acids to proteins by DHHC palmitoylacyl transferases. HTT is palmitoylated by huntingtin interacting proteins 14 and 14-like (HIP14 and HIP14L or ZDHHC17 and 13 respectively). Mutant HTT is less palmitoylated and this reduction of palmitoylation accelerates its aggregation and increases cellular toxicity. Mouse models deficient in either Hip14 (Hip14(-/-)) or Hip14l (Hip14l(-/-)) develop HD-like phenotypes. The biological function of HTT palmitoylation and the role that loss of HTT palmitoylation plays in the pathogenesis of HD are unknown. To address these questions mice deficient for both genes were created. Loss of Hip14 and Hip14l leads to early embryonic lethality at day embryonic day 10-11 due to failed chorioallantoic fusion. The chorion is thickened and disorganized and the allantois does not fuse correctly with the chorion and forms a balloon-like shape compared to Hip14l(-/-); Hip14(+/+) littermate control embryos. Interestingly, the Hip14(-/-) ; Hip14(-/-) embryos share many features with the Htt(-/-) embryos, including folding of the yolk sac, a bulb shaped allantois, and a thickened and disorganized chorion. This may be due to a decrease in HTT palmitoylation. In Hip14(-/-); Hip14l(-/-) mouse embryonic fibroblasts show a 25% decrease in HTT palmitoylation compared to wild type cells. This is the first description of a double PAT deficient mouse model where loss of a PAT or multiple PATs results in embryonic lethality in mammals. These results reinforce the physiological importance of palmitoylation during embryogenesis.

  20. The Biology of Huntingtin.

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    Saudou, Frédéric; Humbert, Sandrine

    2016-03-01

    Huntingtin (HTT) is now a famous protein because an abnormal expansion of a glutamine stretch (polyQ) in its N-terminal sequence leads to the devastating neurodegenerative disorder Huntington's disease (HD). The gene encoding huntingtin, HTT, and its dominantly inherited mutation were identified more than 20 years ago. Subsequently, in the hope of finding a cure for HD, there has been intense research aimed at understanding the molecular mechanisms underlying the deleterious effects of the presence of the abnormal polyQ expansion in HTT. Notwithstanding with the value of this approach, evidence has been emerging of a potential role of context and function of the HTT protein in the specificity and severity of the pathogenicity. HTT is ubiquitous both at the tissue and subcellular levels. It interacts with many partners and has long been considered having no clearly defined cellular function. Based on research over the past 20 years, specifically focused on the function of wild-type HTT, we reconsider the literature describing HTT-regulated molecular and cellular mechanisms that could be dysfunctional in HD and their possible physiological consequences for patients.

  1. Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation*

    Science.gov (United States)

    Zucchelli, Silvia; Marcuzzi, Federica; Codrich, Marta; Agostoni, Elena; Vilotti, Sandra; Biagioli, Marta; Pinto, Milena; Carnemolla, Alisia; Santoro, Claudio; Gustincich, Stefano; Persichetti, Francesca

    2011-01-01

    Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis. PMID:21454471

  2. Tumor necrosis factor receptor-associated factor 6 (TRAF6) associates with huntingtin protein and promotes its atypical ubiquitination to enhance aggregate formation.

    Science.gov (United States)

    Zucchelli, Silvia; Marcuzzi, Federica; Codrich, Marta; Agostoni, Elena; Vilotti, Sandra; Biagioli, Marta; Pinto, Milena; Carnemolla, Alisia; Santoro, Claudio; Gustincich, Stefano; Persichetti, Francesca

    2011-07-15

    Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys(6), Lys(27), and Lys(29) linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys(6), Lys(27), and Lys(29) ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

  3. Effect of post-mortem delay on N-terminal huntingtin protein fragments in human control and Huntington disease brain lysates.

    Science.gov (United States)

    Schut, Menno H; Patassini, Stefano; Kim, Eric H; Bullock, Jocelyn; Waldvogel, Henry J; Faull, Richard L M; Pepers, Barry A; den Dunnen, Johan T; van Ommen, Gert-Jan B; van Roon-Mom, Willeke M C

    2017-01-01

    Huntington disease is associated with elongation of a CAG repeat in the HTT gene that results in a mutant huntingtin protein. Several studies have implicated N-terminal huntingtin protein fragments in Huntington disease pathogenesis. Ideally, these fragments are studied in human brain tissue. However, the use of human brain tissue comes with certain unavoidable variables such as post mortem delay, artefacts from freeze-thaw cycles and subject-to-subject variation. Knowledge on how these variables might affect N-terminal huntingtin protein fragments in post mortem human brain is important for a proper interpretation of study results. The effect of post mortem delay on protein in human brain is known to vary depending on the protein of interest. In the present study, we have assessed the effect of post mortem delay on N-terminal huntingtin protein fragments using western blot. We mimicked post mortem delay in one individual control case and one individual Huntington disease case with low initial post mortem delay. The influence of subject-to-subject variation on N-terminal huntingtin fragments was assessed in human cortex and human striatum using two cohorts of control and Huntington disease subjects. Our results show that effects of post mortem delay on N-terminal huntingtin protein fragments are minor in our individual subjects. Additionally, one freeze-thaw cycle decreases the huntingtin western blot signal intensity in the cortex control subject, but does not introduce additional N-terminal huntingtin fragments. Our results suggest that subject-to-subject variation contributes more to variability in N-terminal huntingtin fragments than post mortem delay.

  4. The role of chaperone-mediated autophagy in huntingtin degradation.

    Directory of Open Access Journals (Sweden)

    Lin Qi

    Full Text Available Huntington Disease (HD is caused by an abnormal expansion of polyQ tract in the protein named huntingtin (Htt. HD pathology is featured by accumulation and aggregation of mutant Htt in striatal and cortical neurons. Aberrant Htt degradation is implicated in HD pathogenesis. The aim of this study was to investigate the regulatory role of chaperone-mediated autophagy (CMA components, heat shock protein cognate 70 (Hsc70 and lysosome-associated protein 2A (LAMP-2A in degradation of Htt fragment 1-552aa (Htt-552. A cell model of HD was produced by overexpression of Htt-552 with adenovirus. The involvement of CMA components in degradation of Htt-552 was determined with over-expression or silencing of Hsc70 and LAMP-2A. The results confirmed previous reports that both macroautophagy and CMA were involved in degradation of Htt-552. Changing the levels of CMA-related proteins affected the accumulation of Htt-552. The lysosomal binding and luminal transport of Htt-552 was demonstrated by incubation of Htt-552 with isolated lysosomes. Expansion of the polyQ tract in Htt-552 impaired its uptake and degradation by lysosomes. Mutation of putative KFERQ motif in wild-type Htt-552 interfered with interactions between Htt-552 and Hsc70. Endogenous Hsc70 and LAMP-2A interacted with exogenously expressed Htt-552. Modulating the levels of CMA related proteins degraded endogenous full-length Htt. These studies suggest that Hsc70 and LAMP-2A through CMA play a role in the clearance of Htt and suggest a novel strategy to target the degradation of mutant Htt.

  5. Neferine attenuates the protein level and toxicity of mutant huntingtin in PC-12 cells via induction of autophagy.

    Science.gov (United States)

    Wong, Vincent Kam Wai; Wu, An Guo; Wang, Jing Rong; Liu, Liang; Law, Betty Yuen-Kwan

    2015-02-18

    Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington's disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74) in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7)-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

  6. Neferine Attenuates the Protein Level and Toxicity of Mutant Huntingtin in PC-12 Cells via Induction of Autophagy

    Directory of Open Access Journals (Sweden)

    Vincent Kam Wai Wong

    2015-02-01

    Full Text Available Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington’s disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74 in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

  7. Huntingtin-associated protein 1 interacts with breakpoint cluster region protein to regulate neuronal differentiation.

    Directory of Open Access Journals (Sweden)

    Pai-Tsang Huang

    Full Text Available Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt-associated protein-1 (Hap1 is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK, resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr, a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.

  8. Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene / allele-specific silencing of mutant huntingtin

    DEFF Research Database (Denmark)

    Carroll, Jeffrey B; Warby, Simon C; Southwell, Amber L;

    2011-01-01

    Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion in the huntingtin gene (HTT) that results in a toxic gain of function in the mutant huntingtin protein (mHTT). Reducing the expression of mHTT is therefore an attractive therapy for HD. However, wild......-type HTT protein is essential for development and has critical roles in maintaining neuronal health. Therapies for HD that reduce wild-type HTT may therefore generate unintended negative consequences. We have identified single-nucleotide polymorphism (SNP) targets in the human HD population for the disease......-specific targeting of the HTT gene. Using primary cells from patients with HD and the transgenic YAC18 and BACHD mouse lines, we developed antisense oligonucleotide (ASO) molecules that potently and selectively silence mHTT at both exonic and intronic SNP sites. Modification of these ASOs with S-constrained-ethyl (c...

  9. Huntingtin interactions with membrane phospholipids: strategic targets for therapeutic intervention?

    Science.gov (United States)

    Kegel-Gleason, Kimberly B

    2013-01-01

    The Huntington's disease gene encodes the protein huntingtin (Htt), a soluble protein that largely distributes to the cytoplasm where about half the protein is found in association with membranes. Early studies on Huntington's disease patients suggested changes in membrane phospholipids. Furthermore, changes in phospholipid biosynthetic enzymes have been found in HD cell models using genetic methods. Recent investigations prove that Htt associates with membranes by direct interactions with phospholipids in membranes. Htt contains at least two membrane binding domains, which may work in concert with each other, to target to the appropriate intracellular membranes for diverse functions. Htt has a particular affinity for a specific class of phospholipids called phosphatidylinositol phosphates; individual species of these phospholipids propagate signals promoting cell survival and regulating changes in morphology. Mutant Htt fragments can disrupt synthetic phospholipid bilayers and full-length mutant Htt shows increased binding to numerous phospholipids, supporting the idea that mutant Htt can introduce pathology at the level of phospholipid interactions. There is a great potential to develop therapeutic agents since numerous enzymes regulate the both the biosynthesis/metabolism of lipids and the post-translational modifications of Htt that direct membrane interactions. Understanding the relationship of Htt with membrane phospholipids, and the impact of mutant Htt on membrane-related functions and lipid metabolism, may help identify new modes of therapeutic intervention for Huntington's disease.

  10. Sequestration of Glyceraldehyde-3-phosphate Dehydrogenase to Aggregates Formed by Mutant Huntingtin

    Institute of Scientific and Technical Information of China (English)

    Junchao WU; Fang LIN; Zhenghong QIN

    2007-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to interact with proteins containing the polyglutamine (polyQ) domain. The present study was undertaken to evaluate the potential contributions of the polyQ and polyproline (polyP) domains to the co-localization of mutant huntingtin (htt) and GAPDH. Overexpression of N-terminal htt (1-969 amino acids) with 100Q and 46Q (htt1-969-100Q and httl-969-46Q, mutant htt) in human mammary gland carcinoma MCF-7 cells formed more htt aggregates than that of htt1-969-18Q (wild-type htt). The co-localization of GAPDH with htt aggregates was found in the cells expressing mutant but not wild-type htt. Deletion of the polyp region in the N-terminal htt had no effect on the co-localization of GAPDH and mutant htt aggregates. These results suggest that the polyQ domain, but not the polyp domain, plays a role in the sequestration of GAPDH to aggregates by mutant htt. This effect might contribute to the dysfunction of neurons caused by mutant htt in Huntington's disease.

  11. Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography*

    Science.gov (United States)

    Darrow, Michele C.; Sergeeva, Oksana A.; Isas, Jose M.; Galaz-Montoya, Jesús G.; King, Jonathan A.; Langen, Ralf; Schmid, Michael F.; Chiu, Wah

    2015-01-01

    Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (∼1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain. PMID:25995452

  12. Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography.

    Science.gov (United States)

    Darrow, Michele C; Sergeeva, Oksana A; Isas, Jose M; Galaz-Montoya, Jesús G; King, Jonathan A; Langen, Ralf; Schmid, Michael F; Chiu, Wah

    2015-07-10

    Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (∼ 1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain.

  13. Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

    OpenAIRE

    Ouyang Xiaosen; Liang Qiuli; Schneider Lonnie; Zhang Jianhua

    2011-01-01

    Abstract Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease a...

  14. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Science.gov (United States)

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington’s disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results. PMID:27110099

  15. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington's disease.

    Science.gov (United States)

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington's disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington's disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson's and Alzheimer's diseases. To identify potential neuroprotective molecules for Huntington's disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington's disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington's disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results.

  16. Normal aging modulates the neurotoxicity of mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Elsa Diguet

    Full Text Available Aging likely plays a role in neurodegenerative disorders. In Huntington's disease (HD, a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt, the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or "normal" aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a beta-Galactosidase (beta-Gal reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week and old (15 month rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that "normal" aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.

  17. Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.

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    Valentina Fodale

    Full Text Available BACKGROUND: In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT, resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT, which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. METHODOLOGY/PRINCIPAL FINDINGS: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein. CONCLUSIONS/SIGNIFICANCE: The temperature- and polyglutamine

  18. Polyglutamine- and Temperature-Dependent Conformational Rigidity in Mutant Huntingtin Revealed by Immunoassays and Circular Dichroism Spectroscopy

    Science.gov (United States)

    Fodale, Valentina; Kegulian, Natalie C.; Verani, Margherita; Cariulo, Cristina; Azzollini, Lucia; Petricca, Lara; Daldin, Manuel; Boggio, Roberto; Padova, Alessandro; Kuhn, Rainer; Pacifici, Robert; Macdonald, Douglas; Schoenfeld, Ryan C.; Park, Hyunsun; Isas, J. Mario; Langen, Ralf; Weiss, Andreas; Caricasole, Andrea

    2014-01-01

    Background In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. Methodology/Principal Findings By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein. Conclusions/Significance The temperature- and polyglutamine-dependent effects

  19. Polyglutamine Amyloid Core Boundaries and Flanking Domain Dynamics in Huntingtin Fragment Fibrils Determined by Solid-State Nuclear Magnetic Resonance

    OpenAIRE

    Hoop, Cody L.; Lin, Hsiang-Kai; Kar, Karunakar; Hou, Zhipeng; Poirier, Michelle A.; Wetzel, Ronald; van der Wel, Patrick C.A.

    2014-01-01

    In Huntington’s disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A “protective” C-terminal proline-rich flanking doma...

  20. Mutant Huntingtin and Elusive Defects in Oxidative Metabolism and Mitochondrial Calcium Handling.

    Science.gov (United States)

    Brustovetsky, Nickolay

    2016-07-01

    Elongation of a polyglutamine (polyQ) stretch in huntingtin protein (Htt) is linked to Huntington's disease (HD) pathogenesis. The mutation in Htt correlates with neuronal dysfunction in the striatum and cerebral cortex and eventually leads to neuronal cell death. The exact mechanisms of the injurious effect of mutant Htt (mHtt) on neurons are not completely understood but might include aberrant gene transcription, defective autophagy, abnormal mitochondrial biogenesis, anomalous mitochondrial dynamics, and trafficking. In addition, deficiency in oxidative metabolism and defects in mitochondrial Ca(2+) handling are considered essential contributing factors to neuronal dysfunction in HD and, consequently, in HD pathogenesis. Since the discovery of the mutation in Htt, the questions whether mHtt affects oxidative metabolism and mitochondrial Ca(2+) handling and, if it does, what mechanisms could be involved were in focus of numerous investigations. However, despite significant research efforts, the detrimental effect of mHtt and the mechanisms by which mHtt might impair oxidative metabolism and mitochondrial Ca(2+) handling remain elusive. In this paper, I will briefly review studies aimed at clarifying the consequences of mHtt interaction with mitochondria and discuss experimental results supporting or arguing against the mHtt effects on oxidative metabolism and mitochondrial Ca(2+) handling.

  1. Autophagy in Huntington disease and huntingtin in autophagy.

    Science.gov (United States)

    Martin, Dale D O; Ladha, Safia; Ehrnhoefer, Dagmar E; Hayden, Michael R

    2015-01-01

    Autophagy is an important biological process that is essential for the removal of damaged organelles and toxic or aggregated proteins by delivering them to the lysosome for degradation. Consequently, autophagy has become a primary target for the treatment of neurodegenerative diseases that involve aggregating proteins. In Huntington disease (HD), an expansion of the polyglutamine (polyQ) tract in the N-terminus of the huntingtin (HTT) protein leads to protein aggregation. However, HD is unique among the neurodegenerative proteinopathies in that autophagy is not only dysfunctional but wild type (wt) HTT also appears to play several roles in regulating the dynamics of autophagy. Herein, we attempt to integrate the recently described novel roles of wtHTT and altered autophagy in HD. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Accelerating the clearance of mutant huntingtin protein aggregates through autophagy induction by europium hydroxide nanorods.

    Science.gov (United States)

    Wei, Peng-Fei; Zhang, Li; Nethi, Susheel Kumar; Barui, Ayan Kumar; Lin, Jun; Zhou, Wei; Shen, Yi; Man, Na; Zhang, Yun-Jiao; Xu, Jing; Patra, Chitta Ranjan; Wen, Long-Ping

    2014-01-01

    Autophagy is one of the well-known pathways to accelerate the clearance of protein aggregates, which contributes to the therapy of neurodegenerative diseases. Although there are numerous reports that demonstrate the induction of autophagy with small molecules including rapamycin, trehalose and lithium, however, there are few reports mentioning the clearance of aggregate-prone proteins through autophagy induction by nanoparticles. In the present article, we have demonstrated that europium hydroxide [Eu(III)(OH)3] nanorods can reduce huntingtin protein aggregation (EGFP-tagged huntingtin protein with 74 polyQ repeats), responsible for neurodegenerative diseases. Again, we have found that these nanorods induce authentic autophagy flux in different cell lines (Neuro 2a, PC12 and HeLa cells) through the expression of higher levels of characteristic autophagy marker protein LC3-II and degradation of selective autophagy substrate/cargo receptor p62/SQSTM1. Furthermore, depression of protein aggregation clearance through the autophagy blockade has also been observed by using specific inhibitors (wortmannin and chloroquine), indicating that autophagy is involved in the degradation of huntingtin protein aggregation. Since [Eu(III)(OH)3] nanorods can enhance the degradation of huntingtin protein aggregation via autophagy induction, we strongly believe that these nanorods would be useful for the development of therapeutic treatment strategies for various neurodegenerative diseases in near future using nanomedicine approach. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. An evolutionary recent neuroepithelial cell adhesion function of huntingtin implicates ADAM10-Ncadherin.

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    Lo Sardo, Valentina; Zuccato, Chiara; Gaudenzi, Germano; Vitali, Barbara; Ramos, Catarina; Tartari, Marzia; Myre, Michael A; Walker, James A; Pistocchi, Anna; Conti, Luciano; Valenza, Marta; Drung, Binia; Schmidt, Boris; Gusella, James; Zeitlin, Scott; Cotelli, Franco; Cattaneo, Elena

    2012-05-01

    The Huntington's disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htt’s ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.

  4. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

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    Bin Huang

    Full Text Available Huntingtin (Htt is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q and mutant (46Q and 128Q Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

  5. Functions of huntingtin in germ layer specification and organogenesis.

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    Giang D Nguyen

    Full Text Available Huntington's disease (HD is a neurodegenerative disease caused by abnormal polyglutamine expansion in the huntingtin protein (Htt. Although both Htt and the HD pathogenic mutation (mHtt are implicated in early developmental events, their individual involvement has not been adequately explored. In order to better define the developmental functions and pathological consequences of the normal and mutant proteins, respectively, we employed embryonic stem cell (ESC expansion, differentiation and induction experiments using huntingtin knock-out (KO and mutant huntingtin knock-in (Q111 mouse ESC lines. In KO ESCs, we observed impairments in the spontaneous specification and survival of ectodermal and mesodermal lineages during embryoid body formation and under inductive conditions using retinoic acid and Wnt3A, respectively. Ablation of BAX improves cell survival, but failed to correct defects in germ layer specification. In addition, we observed ensuing impairments in the specification and maturation of neural, hepatic, pancreatic and cardiomyocyte lineages. These developmental deficits occurred in concert with alterations in Notch, Hes1 and STAT3 signaling pathways. Moreover, in Q111 ESCs, we observed differential developmental stage-specific alterations in lineage specification and maturation. We also observed changes in Notch/STAT3 expression and activation. Our observations underscore essential roles of Htt in the specification of ectoderm, endoderm and mesoderm, in the specification of neural and non-neural organ-specific lineages, as well as cell survival during early embryogenesis. Remarkably, these developmental events are differentially deregulated by mHtt, raising the possibility that HD-associated early developmental impairments may contribute not only to region-specific neurodegeneration, but also to non-neural co-morbidities.

  6. Functions of huntingtin in germ layer specification and organogenesis.

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    Nguyen, Giang D; Molero, Aldrin E; Gokhan, Solen; Mehler, Mark F

    2013-01-01

    Huntington's disease (HD) is a neurodegenerative disease caused by abnormal polyglutamine expansion in the huntingtin protein (Htt). Although both Htt and the HD pathogenic mutation (mHtt) are implicated in early developmental events, their individual involvement has not been adequately explored. In order to better define the developmental functions and pathological consequences of the normal and mutant proteins, respectively, we employed embryonic stem cell (ESC) expansion, differentiation and induction experiments using huntingtin knock-out (KO) and mutant huntingtin knock-in (Q111) mouse ESC lines. In KO ESCs, we observed impairments in the spontaneous specification and survival of ectodermal and mesodermal lineages during embryoid body formation and under inductive conditions using retinoic acid and Wnt3A, respectively. Ablation of BAX improves cell survival, but failed to correct defects in germ layer specification. In addition, we observed ensuing impairments in the specification and maturation of neural, hepatic, pancreatic and cardiomyocyte lineages. These developmental deficits occurred in concert with alterations in Notch, Hes1 and STAT3 signaling pathways. Moreover, in Q111 ESCs, we observed differential developmental stage-specific alterations in lineage specification and maturation. We also observed changes in Notch/STAT3 expression and activation. Our observations underscore essential roles of Htt in the specification of ectoderm, endoderm and mesoderm, in the specification of neural and non-neural organ-specific lineages, as well as cell survival during early embryogenesis. Remarkably, these developmental events are differentially deregulated by mHtt, raising the possibility that HD-associated early developmental impairments may contribute not only to region-specific neurodegeneration, but also to non-neural co-morbidities.

  7. Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

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    Ouyang Xiaosen

    2011-06-01

    Full Text Available Abstract Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity. Results In this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy. Conclusions These observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.

  8. An Expanded CAG Repeat in Huntingtin Causes +1 Frameshifting.

    Science.gov (United States)

    Saffert, Paul; Adamla, Frauke; Schieweck, Rico; Atkins, John F; Ignatova, Zoya

    2016-08-26

    Maintenance of triplet decoding is crucial for the expression of functional protein because deviations either into the -1 or +1 reading frames are often non-functional. We report here that expression of huntingtin (Htt) exon 1 with expanded CAG repeats, implicated in Huntington pathology, undergoes a sporadic +1 frameshift to generate from the CAG repeat a trans-frame AGC repeat-encoded product. This +1 recoding is exclusively detected in pathological Htt variants, i.e. those with expanded repeats with more than 35 consecutive CAG codons. An atypical +1 shift site, UUC C at the 5' end of CAG repeats, which has some resemblance to the influenza A virus shift site, triggers the +1 frameshifting and is enhanced by the increased propensity of the expanded CAG repeats to form a stem-loop structure. The +1 trans-frame-encoded product can directly influence the aggregation of the parental Htt exon 1.

  9. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

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    Zeng YX

    2016-04-01

    Full Text Available Yixuan Zeng,1,2,* Wenyuan Guo,1,* Guangqing Xu,3 Qinmei Wang,4 Luyang Feng,1,2 Simei Long,1 Fengyin Liang,1 Yi Huang,1 Xilin Lu,1 Shichang Li,5 Jiebin Zhou,5 Jean-Marc Burgunder,6 Jiyan Pang,5 Zhong Pei1,2 1Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Disease, The First Affiliated Hospital, Sun Yat-sen University, 2Guangzhou Center, Chinese Huntington’s Disease Network, 3Department of Rehabilitation, The First Affiliated Hospital, 4Key laboratory on Assisted Circulation, Ministry of Health, Department of Cardiovascular Medicine of the First Affiliated Hospital, 5School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; 6Swiss Huntington’s Disease Center, Department of Neurology, University of Bern, Bern, Switzerland *These authors contributed equally to this work Abstract: Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt. Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a

  10. Msh2 acts in medium-spiny striatal neurons as an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington's disease knock-in mice.

    Science.gov (United States)

    Kovalenko, Marina; Dragileva, Ella; St Claire, Jason; Gillis, Tammy; Guide, Jolene R; New, Jaclyn; Dong, Hualing; Kucherlapati, Raju; Kucherlapati, Melanie H; Ehrlich, Michelle E; Lee, Jong-Min; Wheeler, Vanessa C

    2012-01-01

    The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.

  11. Ubiquilin/Dsk2 promotes inclusion body formation and vacuole (lysosome)-mediated disposal of mutated huntingtin.

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    Chuang, Kun-Han; Liang, Fengshan; Higgins, Ryan; Wang, Yanchang

    2016-07-01

    Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant (dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by human ubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins.

  12. Native Mutant Huntingtin in Human Brain

    Science.gov (United States)

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  13. Grb2 is regulated by foxd3 and has roles in preventing accumulation and aggregation of mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Shounak Baksi

    Full Text Available Growth factor receptor protein binding protein 2 (Grb2 is known to be associated with intracellular growth and proliferation related signaling cascades. Huntingtin (Htt, a ubiquitously expressed protein, when mutated, forms toxic intracellular aggregates - the hallmark of Huntington's disease (HD. We observed an elevated expression of Grb2 in neuronal cells in animal and cell models of HD. Grb2 overexpression was predominantly regulated by the transcription factor Forkhead Box D3 (Foxd3. Exogenous expression of Grb2 also reduced aggregation of mutant Htt in Neuro2A cells. Grb2 is also known to interact with Htt, depending on epidermal growth factor receptor (EGFR activation. Grb2- mutant Htt interaction in the contrary, took place in vesicular structures, independent of EGFR activation that eventually merged with autophagosomes and activated the autophagy machinery helping in autophagosome and lysosome fusion. Grb2, with its emerging dual role, holds promise for a survival mechanism for HD.

  14. In vivo proof-of-concept of removal of the huntingtin caspase cleavage motif-encoding exon 12 approach in the YAC128 mouse model of Huntington's disease.

    Science.gov (United States)

    Casaca-Carreira, João; Toonen, Lodewijk J A; Evers, Melvin M; Jahanshahi, Ali; van-Roon-Mom, Willeke M C; Temel, Yasin

    2016-12-01

    Huntington's disease (HD) is a progressive autosomal dominant disease, caused by a CAG repeat expansion in the HTT gene, resulting in an expanded polyglutamine stretch at the N-terminal of the huntingtin protein. An important event in HD pathogenesis appears to be the proteolysis of the mutant protein, which forms N-terminal huntingtin fragments. These fragments form insoluble aggregates and are found in nuclei and cytoplasm of affected neurons where they interfere with normal cell functioning. Important cleavage sites are encoded by exon 12 of HTT. A novel approach is Htt protein modification through exon skipping, which has recently been proven effective both in vitro and in vivo. Here we report proof-of-concept of AON 12.1 in vivo using the YAC128 mouse model of HD. Our results support and encourage future longitudinal studies exploring the therapeutic effects of sustained infusions in the YAC128 mouse model.

  15. Targeting CAG repeat RNAs reduces Huntington’s disease phenotype independently of huntingtin levels

    Science.gov (United States)

    Rué, Laura; Bañez-Coronel, Mónica; Creus-Muncunill, Jordi; Giralt, Albert; Alcalá-Vida, Rafael; Mentxaka, Gartze; Kagerbauer, Birgit; Aranda, Zeus; Venturi, Veronica; Pérez-Navarro, Esther; Estivill, Xavier

    2016-01-01

    Huntington’s disease (HD) is a polyglutamine disorder caused by a CAG expansion in the Huntingtin (HTT) gene exon 1. This expansion encodes a mutant protein whose abnormal function is traditionally associated with HD pathogenesis; however, recent evidence has also linked HD pathogenesis to RNA stable hairpins formed by the mutant HTT expansion. Here, we have shown that a locked nucleic acid–modified antisense oligonucleotide complementary to the CAG repeat (LNA-CTG) preferentially binds to mutant HTT without affecting HTT mRNA or protein levels. LNA-CTGs produced rapid and sustained improvement of motor deficits in an R6/2 mouse HD model that was paralleled by persistent binding of LNA-CTG to the expanded HTT exon 1 transgene. Motor improvement was accompanied by a pronounced recovery in the levels of several striatal neuronal markers severely impaired in R6/2 mice. Furthermore, in R6/2 mice, LNA-CTG blocked several pathogenic mechanisms caused by expanded CAG RNA, including small RNA toxicity and decreased Rn45s expression levels. These results suggest that LNA-CTGs promote neuroprotection by blocking the detrimental activity of CAG repeats within HTT mRNA. The present data emphasize the relevance of expanded CAG RNA to HD pathogenesis, indicate that inhibition of HTT expression is not required to reverse motor deficits, and further suggest a therapeutic potential for LNA-CTG in polyglutamine disorders. PMID:27721240

  16. Polyglutamine expansion in huntingtin increases its insertion into lipid bilayers.

    Science.gov (United States)

    Kegel, Kimberly B; Schewkunow, Vitali; Sapp, Ellen; Masso, Nicholas; Wanker, Erich E; DiFiglia, Marian; Goldmann, Wolfgang H

    2009-09-25

    An expanded polyglutamine (Q) tract (>37Q) in huntingtin (htt) causes Huntington disease. Htt associates with membranes and polyglutamine expansion in htt may alter membrane function in Huntington disease through a mechanism that is not known. Here we used differential scanning calorimetry to examine the effects of polyQ expansion in htt on its insertion into lipid bilayers. We prepared synthetic lipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine and tested interactions of htt amino acids 1-89 with 20Q, 32Q or 53Q with the vesicles. GST-htt1-89 with 53Q inserted into synthetic lipid vesicles significantly more than GST-htt1-89 with 20Q or 32Q. We speculate that by inserting more into cell membranes, mutant huntingtin could increase disorder within the lipid bilayer and thereby disturb cellular membrane function.

  17. Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat

    Science.gov (United States)

    Gagnon, Keith T.; Pendergraff, Hannah M.; Deleavey, Glen F.; Swayze, Eric E.; Potier, Pierre; Randolph, John; Roesch, Eric B.; Chattopadhyaya, Jyoti; Damha, Masad J.; Bennett, C. Frank; Montaillier, Christophe; Lemaitre, Marc; Corey, David R.

    2010-01-01

    Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD. PMID:21028906

  18. Bidirectional control of postsynaptic density-95 (PSD-95) clustering by Huntingtin.

    Science.gov (United States)

    Parsons, Matthew P; Kang, Rujun; Buren, Caodu; Dau, Alejandro; Southwell, Amber L; Doty, Crystal N; Sanders, Shaun S; Hayden, Michael R; Raymond, Lynn A

    2014-02-07

    Huntington disease is associated with early alterations in corticostriatal synaptic function that precede cell death, and it is postulated that ameliorating such changes may delay clinical onset and/or prevent neurodegeneration. Although many of these synaptic alterations are thought to be attributable to a toxic gain of function of the mutant huntingtin protein, the role that nonpathogenic huntingtin (HTT) plays in synaptic function is relatively unexplored. Here, we compare the immunocytochemical localization of a major postsynaptic scaffolding protein, PSD-95, in striatal neurons from WT mice and mice overexpressing HTT with 18 glutamine repeats (YAC18, nonpathogenic). We found that HTT overexpression resulted in a palmitoylation- and BDNF-dependent increase in PSD-95 clustering at synaptic sites in striatal spiny projection neurons (SPNs) co-cultured with cortical neurons. Surprisingly, the latter effect was mediated presynaptically, as HTT overexpression in cortical neurons alone was sufficient to increase PSD-95 clustering in the postsynaptic SPNs. In contrast, antisense oligonucleotide knockdown of HTT in WT co-cultures resulted in a significant reduction of PSD-95 clustering in SPNs. Notably, despite these bidirectional changes in PSD-95 clustering, we did not observe an alteration in basal electrophysiological measures of AMPA and NMDA receptors. Thus, unlike in previous studies in the hippocampus, enhanced or decreased PSD-95 clustering alone was insufficient to drive AMPA or NMDA receptors into or out of SPN synapses. In all, our results demonstrate that nonpathogenic HTT can indeed influence synaptic protein localization and uncover a novel role of HTT in PSD-95 distribution.

  19. Bidirectional Control of Postsynaptic Density-95 (PSD-95) Clustering by Huntingtin*

    Science.gov (United States)

    Parsons, Matthew P.; Kang, Rujun; Buren, Caodu; Dau, Alejandro; Southwell, Amber L.; Doty, Crystal N.; Sanders, Shaun S.; Hayden, Michael R.; Raymond, Lynn A.

    2014-01-01

    Huntington disease is associated with early alterations in corticostriatal synaptic function that precede cell death, and it is postulated that ameliorating such changes may delay clinical onset and/or prevent neurodegeneration. Although many of these synaptic alterations are thought to be attributable to a toxic gain of function of the mutant huntingtin protein, the role that nonpathogenic huntingtin (HTT) plays in synaptic function is relatively unexplored. Here, we compare the immunocytochemical localization of a major postsynaptic scaffolding protein, PSD-95, in striatal neurons from WT mice and mice overexpressing HTT with 18 glutamine repeats (YAC18, nonpathogenic). We found that HTT overexpression resulted in a palmitoylation- and BDNF-dependent increase in PSD-95 clustering at synaptic sites in striatal spiny projection neurons (SPNs) co-cultured with cortical neurons. Surprisingly, the latter effect was mediated presynaptically, as HTT overexpression in cortical neurons alone was sufficient to increase PSD-95 clustering in the postsynaptic SPNs. In contrast, antisense oligonucleotide knockdown of HTT in WT co-cultures resulted in a significant reduction of PSD-95 clustering in SPNs. Notably, despite these bidirectional changes in PSD-95 clustering, we did not observe an alteration in basal electrophysiological measures of AMPA and NMDA receptors. Thus, unlike in previous studies in the hippocampus, enhanced or decreased PSD-95 clustering alone was insufficient to drive AMPA or NMDA receptors into or out of SPN synapses. In all, our results demonstrate that nonpathogenic HTT can indeed influence synaptic protein localization and uncover a novel role of HTT in PSD-95 distribution. PMID:24347167

  20. Mutant huntingtin fragmentation in immune cells tracks Huntington’s disease progression

    OpenAIRE

    Weiss, Andreas; Träger, Ulrike; Wild, Edward J.; Grueninger, Stephan; Farmer, Ruth; Landles, Christian; Scahill, Rachael I.; Lahiri, Nayana; Haider, Salman; Macdonald, Douglas; Frost, Chris; Bates, Gillian P.; Bilbe, Graeme; Kuhn, Rainer; Andre, Ralph

    2012-01-01

    Huntington’s disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Therapeutic approaches to lower mutant HTT (mHTT) levels are expected to proceed to human trials, but noninvasive quantification of mHTT is not currently possible. The importance of the peripheral immune system in neurodegenerative disease is becoming increasingly recognized. Peripheral immune cells have been implicated in HD pathogenesis, but HTT ...

  1. p53 increases caspase-6 expression and activation in muscle tissue expressing mutant huntingtin

    DEFF Research Database (Denmark)

    Ehrnhoefer, Dagmar E; Skotte, Niels H; Ladha, Safia

    2014-01-01

    a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well...... as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse......-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6...

  2. Huntingtin acts non cell-autonomously on hippocampal neurogenesis and controls anxiety-related behaviors in adult mouse.

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    Patrick Pla

    Full Text Available Huntington's disease (HD is a fatal neurodegenerative disease, characterized by motor defects and psychiatric symptoms, including mood disorders such as anxiety and depression. HD is caused by an abnormal polyglutamine (polyQ expansion in the huntingtin (HTT protein. The development and analysis of various mouse models that express pathogenic polyQ-HTT revealed a link between mutant HTT and the development of anxio-depressive behaviors and various hippocampal neurogenesis defects. However, it is unclear whether such phenotype is linked to alteration of HTT wild-type function in adults. Here, we report the analysis of a new mouse model in which HTT is inducibly deleted from adult mature cortical and hippocampal neurons using the CreER(T2/Lox system. These mice present defects in both the survival and the dendritic arborization of hippocampal newborn neurons. Our data suggest that these non-cell autonomous effects are linked to defects in both BDNF transport and release upon HTT silencing in hippocampal neurons, and in BDNF/TrkB signaling. The controlled deletion of HTT also had anxiogenic-like effects. Our results implicate endogenous wild-type HTT in adult hippocampal neurogenesis and in the control of mood disorders.

  3. Characterization of HTT inclusion size, location, and timing in the zQ175 mouse model of Huntington's disease: an in vivo high-content imaging study.

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    Nikisha Carty

    Full Text Available Huntington's disease (HD is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.

  4. Huntingtin-deficient zebrafish exhibit defects in iron utilization and development.

    Science.gov (United States)

    Lumsden, Amanda L; Henshall, Tanya L; Dayan, Sonia; Lardelli, Michael T; Richards, Robert I

    2007-08-15

    Huntington's disease (HD) is one of nine neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine in their respective, otherwise apparently unrelated proteins. Despite these proteins having widespread and overlapping expression patterns in the brain, a specific and unique subset of neurons exhibits particular vulnerability in each disease. It has been hypothesized that perturbation of normal protein function contributes to the specificity of neuronal vulnerability; however, the normal biological functions of many of these proteins including the HD gene product, Huntingtin (Htt), are unclear. To explore the roles of Htt, we have used antisense morpholino oligonucleotides to observe the effects of Htt deficiency in early zebrafish development. Knockdown of Htt expression resulted in a variety of developmental defects. Most notably, Htt-deficient zebrafish had hypochromic blood due to decreased hemoglobin production, despite the presence of iron within blood cells. Furthermore, transferrin receptor 1 transcripts were increased, suggesting cellular iron starvation. Provision of iron to the cytoplasm in a bio-available form restored hemoglobin production in Htt-deficient embryos. Since erythroid cells acquire iron via receptor-mediated endocytosis of transferrin, these results suggest a role for Htt in making endocytosed iron accessible for cellular utilization. Iron is required for oxidative energy production, and defects in iron homeostasis and energy metabolism are features of HD pathogenesis that are most pronounced in the major region of neurodegeneration. It is therefore plausible that perturbation of Htt's normal role in the iron pathway (by polyglutamine tract expansion) contributes to HD pathology, and particularly to its neuronal specificity.

  5. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

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    Wilbur, Jeremy D., E-mail: jwilbur@msg.ucsf.edu [Graduate Program in Biophysics, University of California, San Francisco, California 94143 (United States); Hwang, Peter K. [Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 (United States); Brodsky, Frances M. [The G. W. Hooper Foundation, Departments of Microbiology and Immunology and of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143 (United States); Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 (United States); Fletterick, Robert J. [Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 (United States); Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 (United States); Graduate Program in Biophysics, University of California, San Francisco, California 94143 (United States)

    2010-03-01

    Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

  6. CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice.

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    Apostol, Barbara L; Simmons, Danielle A; Zuccato, Chiara; Illes, Katalin; Pallos, Judit; Casale, Malcolm; Conforti, Paola; Ramos, Catarina; Roarke, Margaret; Kathuria, Satish; Cattaneo, Elena; Marsh, J Lawrence; Thompson, Leslie Michels

    2008-09-01

    Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the protein Huntingtin (Htt). We previously reported that mutant Htt expression activates the ERK1/2 and JNK pathways [Apostol, B.L., Illes, K., Pallos, J., Bodai, L., Wu, J., Strand, A., Schweitzer, E.S., Olson, J.M., Kazantsev, A., Marsh, J.L., Thompson, L.M., 2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 15, 273-285]. Chemical and genetic modulation of these pathways promotes cell survival and death, respectively. Here we test the ability of two closely related compounds, CEP-11004 and CEP-1347, which inhibit Mixed Lineage Kinases (MLKs) and are neuroprotective, to suppress mutant Htt-mediated pathogenesis in multiple model systems. CEP-11004/CEP-1347 treatment significantly decreased toxicity in mutant Htt-expressing cells that evoke a strong JNK response. However, suppression of cellular dysfunction in cell lines that exhibit only mild Htt-associated toxicity and little JNK activation was associated with activation of ERK1/2. These compounds also reduced neurotoxicity in immortalized striatal neurons from mutant knock-in mice and Drosophila expressing a mutant Htt fragment. Finally, CEP-1347 improved motor performance in R6/2 mice and restored expression of BDNF, a critical neurotrophic factor that is reduced in HD. These studies suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via CEP-1347 up-regulation of BDNF.

  7. Huntingtin-Mediated Multipolar-Bipolar Transition of Newborn Cortical Neurons Is Critical for Their Postnatal Neuronal Morphology.

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    Barnat, Monia; Le Friec, Julien; Benstaali, Caroline; Humbert, Sandrine

    2017-01-04

    In the developing cortex, projection neurons undergo multipolar-bipolar transition, radial-directed migration, and maturation. The contribution of these developmental steps to the structure of the adult cortex is not completely understood. Here, we report that huntingtin (HTT), the protein mutated in Huntington's disease, is enriched in polarizing projection neurons. The depletion of HTT in postmitotic projection neurons leads to the mislocalization of layer-specific neuronal populations in the mouse neocortex. HTT is required for the multipolar-bipolar transition of projection neurons and for the maintenance of their bipolar shape during their radial migration. HTT mediates these effects in vivo through the regulation of RAB11-dependent N-Cadherin trafficking. Importantly, HD pathological HTT alters RAB11-dependent neuronal migration. Finally, we show that the cortical defects resulting from the postmitotic loss of HTT specifically during embryonic development affect neuronal morphology at adulthood. Our data reveal a new HTT-RAB11-N-Cadherin pathway regulating multipolar-bipolar transition with direct implications for mature brain. VIDEO ABSTRACT.

  8. The Interaction of Polyglutamine Peptides with Lipid Membranes Is Regulated by Flanking Sequences Associated with Huntingtin*

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    Burke, Kathleen A.; Kauffman, Karlina J.; Umbaugh, C. Samuel; Frey, Shelli L.; Legleiter, Justin

    2013-01-01

    Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain. PMID:23572526

  9. A series of N-terminal epitope tagged Hdh knock-in alleles expressing normal and mutant huntingtin: their application to understanding the effect of increasing the length of normal huntingtin’s polyglutamine stretch on CAG140 mouse model pathogenesis

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    Zheng Shuqiu

    2012-08-01

    Full Text Available Abstract Background Huntington’s disease (HD is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ stretch within Huntingtin (htt, the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR in modulating HD mouse model pathogenesis is currently unknown. Results We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh encoding N-terminal hemaglutinin (HA or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt. Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt’s polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis. Conclusion In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes.

  10. Msh2 acts in medium-spiny striatal neurons as an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington's disease knock-in mice.

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    Marina Kovalenko

    Full Text Available The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111 with mice carrying a conditional (floxed Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.

  11. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

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    Tebbenkamp, Andrew T N; Crosby, Keith W; Siemienski, Zoe B; Brown, Hilda H; Golde, Todd E; Borchelt, David R

    2012-01-01

    N-terminal fragments of mutant huntingtin (htt) that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1), form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD). These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments. Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like), were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1. Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

  12. Wild-type huntingtin ameliorates striatal neuronal atrophy but does not prevent other abnormalities in the YAC128 mouse model of Huntington disease

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    Leavitt Blair R

    2006-12-01

    Full Text Available Abstract Background Huntington disease (HD is an adult onset neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (htt protein. Htt function is essential for embryonic survival as well as normal function during the postnatal period. In addition to having roles in transcription and transport, recent evidence demonstrates that wild-type htt is neuroprotective in vivo. To determine whether treatment with wild-type htt would be beneficial in HD, we crossed the YAC128 mouse model of HD with mice that over-express wild-type htt (YAC18 mice to generate YAC128 mice that over-express wild-type htt (YAC18/128 mice. Results YAC18/128 mice were found to express mutant htt at the same level as YAC128 mice and wild-type htt at the same level as YAC18 mice. YAC18/128 mice show no significant behavioural improvement compared to YAC128 mice in the rotarod test of motor coordination or in an automated open field test. In the brain, YAC18/128 mice show no significant improvement in striatal volume, striatal neuronal numbers or striatal DARPP-32 expression compared to YAC128 mice. In contrast, striatal neuronal cross-sectional area showed significant improvement in YAC18/128 mice compared to YAC128 mice. Conclusion While the over-expression of wild-type htt results in a mild improvement in striatal neuropathology in YAC128 mice, our findings suggest that treatment with wild-type htt may not be sufficient to ameliorate the symptoms of HD in this model.

  13. Differential effect of HDAC3 on cytoplasmic and nuclear huntingtin aggregates.

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    Tatsuo Mano

    Full Text Available Histone deacetylases (HDACs are potential therapeutic targets of polyglutamine (pQ diseases including Huntington's disease (HD that may function to correct aberrant transcriptional deactivation caused by mutant pQ proteins. HDAC3 is a unique class 1 HDAC found in both the cytoplasm and in the nucleus. However, the precise functions of HDAC3 in the two cellular compartments are only vaguely known. HDAC3 directly binds to huntingtin (Htt with short pQ and this interaction is important for suppressing neurotoxicity induced by HDAC3. With long pQ Htt, the interaction with HDAC3 is inhibited, and this supposedly promotes neuronal death, indicating that HDAC3 would be a good therapeutic target for HD. However, the knockout of one HDAC3 allele did not show any efficacy in reducing neurodegenerative symptoms in a mouse model of HD. Therefore, the role of HDAC3 in the pathogenesis of HD has yet to be fully elucidated. We attempted to resolve this issue by focusing on the different roles of HDAC3 on cytoplasmic and nuclear Htt aggregates. In addition to supporting the previous findings, we found that HDAC3 preferentially binds to nuclear Htt over cytoplasmic ones. Specific HDAC3 inhibitors increased the total amount of Htt aggregates by increasing the amount of nuclear aggregates. Both cytoplasmic and nuclear Htt aggregates were able to suppress endogenous HDAC3 activity, which led to decreased nuclear proteasome activity. Therefore, we concluded that Htt aggregates impair nuclear proteasome activity through the inhibition of HDAC3. Our findings provide new insights regarding cross-compartment proteasome regulation.

  14. Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer.

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    Bankanidhi Sahoo

    Full Text Available Expansion of the polyglutamine (polyQ track of the Huntingtin (HTT protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD. Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the

  15. Huntington's disease and mitochondrial DNA deletions: event or regular mechanism for mutant huntingtin protein and CAG repeats expansion?!

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    Banoei, Mohammad Mehdi; Houshmand, Massoud; Panahi, Mehdi Shafa Shariat; Shariati, Parvin; Rostami, Maryam; Manshadi, Masoumeh Dehghan; Majidizadeh, Tayebeh

    2007-11-01

    The mitochondrial DNA (mtDNA) may play an essential role in the pathogenesis of the respiratory chain complex activities in neurodegenerative disorders such as Huntington's disease (HD). Research studies were conducted to determine the possible levels of mitochondrial defect (deletion) in HD patients and consideration of interaction between the expanded Huntingtin gene as a nuclear gene and mitochondria as a cytoplasmic organelle. To determine mtDNA damage, we investigated deletions based in four areas of mitochondrial DNA, in a group of 60 Iranian patients clinically diagnosed with HD and 70 healthy controls. A total of 41 patients out of 60 had CAG expansion (group A). About 19 patients did not show expansion but had the clinical symptoms of HD (group B). MtDNA deletions were classified into four groups according to size; 9 kb, 7.5 kb, 7 kb, and 5 kb. We found one of the four-mtDNA deletions in at least 90% of samples. Multiple deletions have also been observed in 63% of HD patients. None of the normal control (group C) showed mtDNA deletions. The sizes or locations of the deletions did not show a clear correlation with expanded CAG repeat and age in our samples. The study presented evidence that HD patients had higher frequencies of mtDNA deletions in lymphocytes in comparison to the controls. It is thus proposed that CAG repeats instability and mutant Htt are causative factor in mtDNA damage.

  16. Behavioral analysis of the huntingtin-associated protein 1 ortholog trak-1 in Caenorhabditis elegans.

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    Norflus, Fran; Bu, Jingnan; Guyton, Evon; Gutekunst, Claire-Anne

    2016-09-01

    The precise role of huntingtin-associated protein 1 (HAP1) is not known, but studies have shown that it is important for early development and survival. A Caenorhabditis elegans ortholog of HAP1, T27A3.1 (also called trak-1), has been found and is expressed in a subset of neurons. Potential behavioral functions of three knockout lines of T27A3.1 were examined. From its suspected role in mice we hypothesize that T27A3.1 might be involved in egg hatching and early growth, mechanosensation, chemosensation, sensitivity to osmolarity, and synaptic transmission. Our studies show that the knockout worms are significantly different from the wild-type (WT) worms only in the synaptic transmission test, which was measured by adding aldicarb, an acetylcholinesterase inhibitor. The change in function was determined by measuring the number of worms paralyzed. However, when the T27A3.1 worms were tested for egg hatching and early growth, mechanosensation, chemosensation, and sensitivity to osmolarity, there were no significant differences between the knockout and WT worms. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Effects of deletion of mutant huntingtin in steroidogenic factor 1 neurons on the psychiatric and metabolic phenotype in the BACHD mouse model of Huntington disease.

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    Barbara Baldo

    Full Text Available Psychiatric and metabolic features appear several years before motor disturbances in the neurodegenerative Huntington's disease (HD, caused by an expanded CAG repeat in the huntingtin (HTT gene. Although the mechanisms leading to these aspects are unknown, dysfunction in the hypothalamus, a brain region controlling emotion and metabolism, has been suggested. A direct link between the expression of the disease causing protein, huntingtin (HTT, in the hypothalamus and the development of metabolic and psychiatric-like features have been shown in the BACHD mouse model of HD. However, precisely which circuitry in the hypothalamus is critical for these features is not known. We hypothesized that expression of mutant HTT in the ventromedial hypothalamus, an area involved in the regulation of metabolism and emotion would be important for the development of these non-motor aspects. Therefore, we inactivated mutant HTT in a specific neuronal population of the ventromedial hypothalamus expressing the transcription factor steroidogenic factor 1 (SF1 in the BACHD mouse using cross-breeding based on a Cre-loxP system. Effects on anxiety-like behavior were assessed using the elevated plus maze and novelty-induced suppressed feeding test. Depressive-like behavior was assessed using the Porsolt forced swim test. Effects on the metabolic phenotype were analyzed using measurements of body weight and body fat, as well as serum insulin and leptin levels. Interestingly, the inactivation of mutant HTT in SF1-expressing neurons exerted a partial positive effect on the depressive-like behavior in female BACHD mice at 4 months of age. In this cohort of mice, no anxiety-like behavior was detected. The deletion of mutant HTT in SF1 neurons did not have any effect on the development of metabolic features in BACHD mice. Taken together, our results indicate that mutant HTT regulates metabolic networks by affecting hypothalamic circuitries that do not involve the SF1 neurons

  18. Huntingtin processing in pathogenesis of Huntington disease

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    Zhenghong QIN; Zhenlun GU

    2004-01-01

    Huntington's disease (HD) is caused by an expansion of the polyglutamine tract in the protein named huntingtin.The expansion of polyglutamine tract induces selective degeneration of striatal projection neurons and cortical pyramidal neurons. The bio-hallmark of HD is the formation of intranuclear inclusions and cytoplasmic aggregates in association with other cellular proteins in vulnerable neurons. Accumulation of N-terminal mutant huntingtin in HD brains is prominent. These pathological features are related to protein misfolding and impairments in protein processing and degradation in neurons. This review focused on the role of proteases in huntingtin cleavage and degradation and the contribution of altered processing of mutant huntingtin to HD pathogenesis.

  19. AAV-mediated delivery of the transcription factor XBP1s into the striatum reduces mutant Huntingtin aggregation in a mouse model of Huntington's disease

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    Zuleta, Amparo [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago (Chile); Vidal, Rene L. [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Neurounion Biomedical Foundation, Santiago (Chile); Armentano, Donna; Parsons, Geoffrey [Department of Molecular Biology, Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Hetz, Claudio, E-mail: chetz@med.uchile.cl [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago (Chile); Department of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Av., Boston, MA 02446 (United States); Neurounion Biomedical Foundation, Santiago (Chile)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer The contribution of ER stress to HD has not been directly addressed. Black-Right-Pointing-Pointer Expression of XBP1s using AAVs decreases Huntingtin aggregation in vivo. Black-Right-Pointing-Pointer We describe a new in vivo model of HD based on the expression of a large fragment of mHtt-RFP. -- Abstract: Huntington's disease (HD) is caused by mutations that expand a polyglutamine region in the amino-terminal domain of Huntingtin (Htt), leading to the accumulation of intracellular inclusions and progressive neurodegeneration. Recent reports indicate the engagement of endoplasmic reticulum (ER) stress responses in human HD post mortem samples and animal models of the disease. Adaptation to ER stress is mediated by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that attenuates protein folding stress by controlling the expression of distinct transcription factors including X-Box binding protein 1 (XBP1). Here we targeted the expression of XBP1 on a novel viral-based model of HD. We delivered an active form of XBP1 locally into the striatum of adult mice using adeno-associated vectors (AAVs) and co-expressed this factor with a large fragment of mutant Htt as a fusion protein with RFP (Htt588{sup Q95}-mRFP) to directly visualize the accumulation of Htt inclusions in the brain. Using this approach, we observed a significant reduction in the accumulation of Htt588{sup Q95}-mRFP intracellular inclusion when XBP1 was co-expressed in the striatum. These results contrast with recent findings indicating a protective effect of XBP1 deficiency in neurodegeneration using knockout mice, and suggest a potential use of gene therapy strategies to manipulate the UPR in the context of HD.

  20. Selective roles of normal and mutant huntingtin in neural induction and early neurogenesis.

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    Nguyen, Giang D; Gokhan, Solen; Molero, Aldrin E; Mehler, Mark F

    2013-01-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal polyglutamine expansion in the amino-terminal end of the huntingtin protein (Htt) and characterized by progressive striatal and cortical pathology. Previous reports have shown that Htt is essential for embryogenesis, and a recent study by our group revealed that the pathogenic form of Htt (mHtt) causes impairments in multiple stages of striatal development. In this study, we have examined whether HD-associated striatal developmental deficits are reflective of earlier maturational alterations occurring at the time of neurulation by assessing differential roles of Htt and mHtt during neural induction and early neurogenesis using an in vitro mouse embryonic stem cell (ESC) clonal assay system. We demonstrated that the loss of Htt in ESCs (KO ESCs) severely disrupts the specification of primitive and definitive neural stem cells (pNSCs, dNSCs, respectively) during the process of neural induction. In addition, clonally derived KO pNSCs and dNSCs displayed impaired proliferative potential, enhanced cell death and altered multi-lineage potential. Conversely, as observed in HD knock-in ESCs (Q111 ESCs), mHtt enhanced the number and size of pNSC clones, which exhibited enhanced proliferative potential and precocious neuronal differentiation. The transition from Q111 pNSCs to fibroblast growth factor 2 (FGF2)-responsive dNSCs was marked by potentiation in the number of dNSCs and altered proliferative potential. The multi-lineage potential of Q111 dNSCs was also enhanced with precocious neurogenesis and oligodendrocyte progenitor elaboration. The generation of Q111 epidermal growth factor (EGF)-responsive dNSCs was also compromised, whereas their multi-lineage potential was unaltered. These abnormalities in neural induction were associated with differential alterations in the expression profiles of Notch, Hes1 and Hes5. These cumulative observations indicate that Htt is required for multiple stages

  1. Selective roles of normal and mutant huntingtin in neural induction and early neurogenesis.

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    Giang D Nguyen

    Full Text Available Huntington's disease (HD is a neurodegenerative disorder caused by abnormal polyglutamine expansion in the amino-terminal end of the huntingtin protein (Htt and characterized by progressive striatal and cortical pathology. Previous reports have shown that Htt is essential for embryogenesis, and a recent study by our group revealed that the pathogenic form of Htt (mHtt causes impairments in multiple stages of striatal development. In this study, we have examined whether HD-associated striatal developmental deficits are reflective of earlier maturational alterations occurring at the time of neurulation by assessing differential roles of Htt and mHtt during neural induction and early neurogenesis using an in vitro mouse embryonic stem cell (ESC clonal assay system. We demonstrated that the loss of Htt in ESCs (KO ESCs severely disrupts the specification of primitive and definitive neural stem cells (pNSCs, dNSCs, respectively during the process of neural induction. In addition, clonally derived KO pNSCs and dNSCs displayed impaired proliferative potential, enhanced cell death and altered multi-lineage potential. Conversely, as observed in HD knock-in ESCs (Q111 ESCs, mHtt enhanced the number and size of pNSC clones, which exhibited enhanced proliferative potential and precocious neuronal differentiation. The transition from Q111 pNSCs to fibroblast growth factor 2 (FGF2-responsive dNSCs was marked by potentiation in the number of dNSCs and altered proliferative potential. The multi-lineage potential of Q111 dNSCs was also enhanced with precocious neurogenesis and oligodendrocyte progenitor elaboration. The generation of Q111 epidermal growth factor (EGF-responsive dNSCs was also compromised, whereas their multi-lineage potential was unaltered. These abnormalities in neural induction were associated with differential alterations in the expression profiles of Notch, Hes1 and Hes5. These cumulative observations indicate that Htt is required for

  2. ATRX induction by mutant huntingtin via Cdx2 modulates heterochromatin condensation and pathology in Huntington's disease.

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    Lee, J; Hong, Y K; Jeon, G S; Hwang, Y J; Kim, K Y; Seong, K H; Jung, M-K; Picketts, D J; Kowall, N W; Cho, K S; Ryu, H

    2012-07-01

    Aberrant chromatin remodeling is involved in the pathogenesis of Huntington's disease (HD) but the mechanism is not known. Herein, we report that mutant huntingtin (mtHtt) induces the transcription of alpha thalassemia/mental retardation X linked (ATRX), an ATPase/helicase and SWI/SNF-like chromatin remodeling protein via Cdx-2 activation. ATRX expression was elevated in both a cell line model and transgenic model of HD, and Cdx-2 occupancy of the ATRX promoter was increased in HD. Induction of ATRX expanded the size of promyelocytic leukemia nuclear body (PML-NB) and increased trimethylation of H3K9 (H3K9me3) and condensation of pericentromeric heterochromatin, while knockdown of ATRX decreased PML-NB and H3K9me3 levels. Knockdown of ATRX/dXNP improved the hatch rate of fly embryos expressing mtHtt (Q127). ATRX/dXNP overexpression exacerbated eye degeneration of eye-specific mtHtt (Q127) expressing flies. Our findings suggest that transcriptional alteration of ATRX by mtHtt is involved in pericentromeric heterochromatin condensation and contributes to the pathogenesis of HD.

  3. Human mutant huntingtin disrupts vocal learning in transgenic songbirds.

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    Liu, Wan-Chun; Kohn, Jessica; Szwed, Sarah K; Pariser, Eben; Sepe, Sharon; Haripal, Bhagwattie; Oshimori, Naoki; Marsala, Martin; Miyanohara, Atsushi; Lee, Ramee

    2015-11-01

    Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

  4. Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease.

    Science.gov (United States)

    Yin, Xiangling; Manczak, Maria; Reddy, P Hemachandra

    2016-05-01

    The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

    Science.gov (United States)

    Lopes, Carla; Aubert, Sophie; Bourgois-Rocha, Fany; Barnat, Monia; Rego, Ana Cristina; Déglon, Nicole; Perrier, Anselme L; Humbert, Sandrine

    2016-01-01

    Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

  6. Effects on murine behavior and lifespan of selectively decreasing expression of mutant huntingtin allele by supt4h knockdown.

    Directory of Open Access Journals (Sweden)

    Hui-Min Cheng

    2015-03-01

    Full Text Available Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington's disease (HD and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (Htt alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human HTT alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2'-O-methoxyethyl oligonucleotides (ASOs directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

  7. Effects of CAG repeat length, HTT protein length and protein context on cerebral metabolism measured using magnetic resonance spectroscopy in transgenic mouse models of Huntington's disease.

    Science.gov (United States)

    Jenkins, Bruce G; Andreassen, Ole A; Dedeoglu, Alpaslan; Leavitt, Blair; Hayden, Michael; Borchelt, David; Ross, Christopher A; Ferrante, Robert J; Beal, M Flint

    2005-10-01

    Huntington's disease is a neurodegenerative illness caused by expansion of CAG repeats at the N-terminal end of the protein huntingtin. We examined longitudinal changes in brain metabolite levels using in vivo magnetic resonance spectroscopy in five different mouse models. There was a large (>50%) exponential decrease in N-acetyl aspartate (NAA) with time in both striatum and cortex in mice with 150 CAG repeats (R6/2 strain). There was a linear decrease restricted to striatum in N171-82Q mice with 82 CAG repeats. Both the exponential and linear decreases of NAA were paralleled in time by decreases in neuronal area measured histologically. Yeast artificial chromosome transgenic mice with 72 CAG repeats, but low expression levels, had less striatal NAA loss than the N171-82Q mice (15% vs. 43%). We evaluated the effect of gene context in mice with an approximate 146 CAG repeat on the hypoxanthine phosphoribosyltransferase gene (HPRT). HPRT mice developed an obese phenotype in contrast to weight loss in the R6/2 and N171-82Q mice. These mice showed a small striatal NAA loss (21%), and a possible increase in brain lipids detectable by magnetic resonance (MR) spectroscopy and decreased brain water T1. Our results indicate profound metabolic defects that are strongly affected by CAG repeat length, as well as gene expression levels and protein context.

  8. Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and beta-cell apoptosis.

    Science.gov (United States)

    Berchtold, Lukas Adrian; Størling, Zenia Marian; Ortis, Fernanda; Lage, Kasper; Bang-Berthelsen, Claus; Bergholdt, Regine; Hald, Jacob; Brorsson, Caroline Anna; Eizirik, Decio Laks; Pociot, Flemming; Brunak, Søren; Størling, Joachim

    2011-09-13

    Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing genes in T1D, we performed an in silico "phenome-interactome analysis" on a genome-wide linkage scan dataset. This method prioritizes candidates according to their physical interactions at the protein level with other proteins involved in diabetes. A total of 11 genes were predicted to be likely disease genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17. Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi knockdown experiments established that HIP14 is an antiapoptotic protein required for β-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1β and IFN-γ) that mediate β-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated rat and human islets. Overexpression of HIP14 was associated with a decrease in IL-1β-induced NF-κB activity and protection against IL-1β-mediated apoptosis. Our study demonstrates that the current network biology approach is a valid method to identify genes of importance for T1D and may therefore embody the basis for more rational and targeted therapeutic approaches.

  9. ROCK-phosphorylated vimentin modifies mutant huntingtin aggregation via sequestration of IRBIT

    Directory of Open Access Journals (Sweden)

    Bauer Peter O

    2012-08-01

    Full Text Available Abstract Background Huntington's Disease (HD is a fatal hereditary neurodegenerative disease caused by the accumulation of mutant huntingtin protein (Htt containing an expanded polyglutamine (polyQ tract. Activation of the channel responsible for the inositol-induced Ca2+ release from ensoplasmic reticulum (ER, was found to contribute substantially to neurodegeneration in HD. Importantly, chemical and genetic inhibition of inositol 1,4,5-trisphosphate (IP3 receptor type 1 (IP3R1 has been shown to reduce mutant Htt aggregation. Results In this study, we propose a novel regulatory mechanism of IP3R1 activity by type III intermediate filament vimentin which sequesters the negative regulator of IP3R1, IRBIT, into perinuclear inclusions, and reduces its interaction with IP3R1 resulting in promotion of mutant Htt aggregation. Proteasome inhibitor MG132, which causes polyQ proteins accumulation and aggregation, enhanced the sequestration of IRBIT. Furthermore we found that IRBIT sequestration can be prevented by a rho kinase inhibitor, Y-27632. Conclusions Our results suggest that vimentin represents a novel and additional target for the therapy of polyQ diseases.

  10. Structural perturbations on huntingtin N17 domain during its folding on 2D-nanomaterials

    Science.gov (United States)

    Zhang, Leili; Feng, Mei; Zhou, Ruhong; Luan, Binquan

    2017-09-01

    A globular protein’s folded structure in its physiological environment is largely determined by its amino acid sequence. Recently, newly discovered transformer proteins as well as intrinsically disordered proteins may adopt the folding-upon-binding mechanism where their secondary structures are highly dependent on their binding partners. Due to the various applications of nanomaterials in biological sensors and potential wearable devices, it is important to discover possible conformational changes of proteins on nanomaterials. Here, through molecular dynamics simulations, we show that the first 17 residues of the huntingtin protein (HTT-N17) exhibit appreciable differences during its folding on 2D-nanomaterials, such as graphene and MoS2 nanosheets. Namely, the protein is disordered on the graphene surface but is helical on the MoS2 surface. Despite that the amphiphilic environment at the nanosheet-water interface promotes the folding of the amphipathic proteins (such as HTT-N17), competitions between protein-nanosheet and intra-protein interactions yield very different protein conformations. Therefore, as engineered binding partners, nanomaterials might significantly affect the structures of adsorbed proteins.

  11. Huntingtin-associated protein 1 (HAP1) interacts with androgen receptor (AR) and suppresses SBMA-mutant-AR-induced apoptosis.

    Science.gov (United States)

    Takeshita, Yukio; Fujinaga, Ryutaro; Zhao, Changjiu; Yanai, Akie; Shinoda, Koh

    2006-08-01

    Huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, has been known as an essential component of the stigmoid body (STB) and recently reported to play a protective role against neurodegeneration in Huntington's disease (HD). In the present study, subcellular association between HAP1 and androgen receptor (AR) with a long polyglutamine tract (polyQ) derived from spinal-and-bulbar-muscular-atrophy (SBMA) was examined using HEp-2 cells cotransfected with HAP1 and/or normal ARQ25, SBMA-mutant ARQ65 or deletion-mutant AR cDNAs. The results provided the first clear evidence that HAP1 interacts with AR through its ligand-binding domain in a polyQ-length-dependent manner and forms prominent inclusions sequestering polyQ-AR, and that addition of dihydrotestosterone reduces the association strength of HAP1 with ARQ25 more dramatically than that with ARQ65. Furthermore, SBMA-mutant-ARQ65-induced apoptosis was suppressed by cotransfection with HAP1. Our findings strongly suggest that HAP1/STB is relevant to polyQ-length-dependent modification on subcellular AR functions and critically involved in pathogenesis of not only HD but also SBMA as an important intrinsic neuroprotectant determining the threshold for cellular vulnerability to apoptosis. Taking together with previous reports that HAP1/STB is selectively expressed in the brain regions spared from degenerative targets in HD and SBMA, the current study might explain the region-specific occurrence of neurodegeneration in both diseases, shedding light on common aspects of their molecular pathological mechanism and yet-to-be-uncovered diagnostic or therapeutic applications for HD and SBMA patients.

  12. Structure of full-length ubiquitin-conjugating enzyme E2-25K (huntingtin-interacting protein 2)

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Randall C.; Hughes, Ronny C.; Flatt, Justin W.; Meehan, Edward J.; Ng, Joseph D.; Twigg, Pamela D.; (UAH)

    2009-08-07

    The ubiquitin-conjugating enzyme E2-25K has been identified as a huntingtin (the key protein in Huntington's disease) interacting protein and has been shown to play a role in mediating the toxicity of A{beta}, the principal protein involved in Alzheimer's disease pathogenesis. E2-25K is a dual-domain protein with an ubiquitin-associated (UBA) domain as well as a conserved ubiquitin-conjugating (UBC) domain which catalyzes the formation of a covalent bond between the C-terminal glycine of an ubiquitin molecule and the {var_epsilon}-amine of a lysine residue on the acceptor protein as part of the ubiquitin-proteasome pathway. The crystal structures of E2-25K M172A mutant protein at pH 6.5 and pH 8.5 were determined to 1.9 and 2.2 {angstrom} resolution, respectively. Examination of the structures revealed domain-domain interactions between the UBC and UBA domains which have not previously been reported

  13. Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.

    Science.gov (United States)

    Langfelder, Peter; Cantle, Jeffrey P; Chatzopoulou, Doxa; Wang, Nan; Gao, Fuying; Al-Ramahi, Ismael; Lu, Xiao-Hong; Ramos, Eliana Marisa; El-Zein, Karla; Zhao, Yining; Deverasetty, Sandeep; Tebbe, Andreas; Schaab, Christoph; Lavery, Daniel J; Howland, David; Kwak, Seung; Botas, Juan; Aaronson, Jeffrey S; Rosinski, Jim; Coppola, Giovanni; Horvath, Steve; Yang, X William

    2016-04-01

    To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo.

  14. Investigating the Structural Impact of the Glutamine Repeat in Huntingtin Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Perevozchikova, Tatiana [ORNL; Stanley, Christopher B [ORNL; McWilliams-Koeppen, Helen P [ORNL; Rowe, Erica L [ORNL; Berthelier-Jung, Valerie M [ORNL

    2014-01-01

    Acquiring detailed structural information about the various aggregation states of the huntingtin-exon1 protein (Htt-exon1) is crucial not only for identifying the true nature of the neurotoxic species responsible for Huntington s disease (HD) but also for designing effective therapeutics. Using time-resolved small-angle neutron scattering (TR-SANS), we followed the conformational changes that occurred during fibrillization of the pathologic form of Htt-exon1 (NtQ42P10) and compared the results with those obtained for the wild-type (NtQ22P10). Our results show that the aggregation pathway of NtQ22P10 is very different from that of NtQ42P10, as the initial steps require a monomer to 7-mer transition stage. In contrast, the earliest species identified for NtQ42P10 are monomer and dimer. The divergent pathways ultimately result in NtQ22P10 fibrils that possess a pack- ing arrangement consistent with the common amyloid sterical zipper model, whereas NtQ42P10 fibrils present a better fit to the Perutz b-helix structural model. The structural details obtained by TR-SANS should help to delineate the key mechanisms that underpin Htt-exon1 aggregation leading to HD.

  15. Dysregulated striatal neuronal processing and impaired motor behavior in mice lacking huntingtin interacting protein 14 (HIP14.

    Directory of Open Access Journals (Sweden)

    Ana María Estrada-Sánchez

    Full Text Available Palmitoyl acyl transferases (PATs play a critical role in protein trafficking and function. Huntingtin interacting protein 14 (HIP14 is a PAT that acts on proteins associated with neuronal transmission, suggesting that deficient protein palmitoylation by HIP14, which occurs in the YAC128 model of Huntington's disease (HD, might have deleterious effects on neurobehavioral processing. HIP14 knockout mice show biochemical and neuropathological changes in the striatum, a forebrain region affected by HD that guides behavioral choice and motor flexibility. Thus, we evaluated the performance of these mice in two tests of motor ability: nest-building and plus maze turning behavior. Relative to wild-type controls, HIP14 knockout mice show impaired nest building and decreased turning in the plus maze. When we recorded the activity of striatal neurons during plus-maze performance, we found faster firing rates and dysregulated spike bursting in HIP14 knockouts compared to wild-type. There was also less correlated firing between simultaneously recorded neuronal pairs in the HIP14 knockouts. Overall, our results indicate that HIP14 is critically involved in behavioral modulation of striatal processing. In the absence of HIP14, striatal neurons become dysfunctional, leading to impaired motor behavior.

  16. Huntingtin Regulates Mammary Stem Cell Division and Differentiation

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    Salah Elias

    2014-04-01

    Full Text Available Little is known about the mechanisms of mitotic spindle orientation during mammary gland morphogenesis. Here, we report the presence of huntingtin, the protein mutated in Huntington’s disease, in mouse mammary basal and luminal cells throughout mammogenesis. Keratin 5-driven depletion of huntingtin results in a decreased pool and specification of basal and luminal progenitors, and altered mammary morphogenesis. Analysis of mitosis in huntingtin-depleted basal progenitors reveals mitotic spindle misorientation. In mammary cell culture, huntingtin regulates spindle orientation in a dynein-dependent manner. Huntingtin is targeted to spindle poles through its interaction with dynein and promotes the accumulation of NUMA and LGN. Huntingtin is also essential for the cortical localization of dynein, dynactin, NUMA, and LGN by regulating their kinesin 1-dependent trafficking along astral microtubules. We thus suggest that huntingtin is a component of the pathway regulating the orientation of mammary stem cell division, with potential implications for their self-renewal and differentiation properties.

  17. HTT-lowering reverses Huntington's disease immune dysfunction caused by NFκB pathway dysregulation.

    Science.gov (United States)

    Träger, Ulrike; Andre, Ralph; Lahiri, Nayana; Magnusson-Lind, Anna; Weiss, Andreas; Grueninger, Stephan; McKinnon, Chris; Sirinathsinghji, Eva; Kahlon, Shira; Pfister, Edith L; Moser, Roger; Hummerich, Holger; Antoniou, Michael; Bates, Gillian P; Luthi-Carter, Ruth; Lowdell, Mark W; Björkqvist, Maria; Ostroff, Gary R; Aronin, Neil; Tabrizi, Sarah J

    2014-03-01

    Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington's disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington's disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington's disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington's disease.

  18. Abnormal mitochondrial dynamics, mitochondrial loss and mutant huntingtin oligomers in Huntington's disease: implications for selective neuronal damage.

    Science.gov (United States)

    Shirendeb, Ulziibat; Reddy, Arubala P; Manczak, Maria; Calkins, Marcus J; Mao, Peizhong; Tagle, Danilo A; Reddy, P Hemachandra

    2011-04-01

    The purpose of our study was to determine the relationship between mutant huntingtin (Htt) and mitochondrial dynamics in the progression of Huntington's disease (HD). We measured the mRNA levels of electron transport chain genes, and mitochondrial structural genes, Drp1 (dynamin-related protein 1), Fis1 (fission 1), Mfn1 (mitofusin 1), Mfn2 (mitofusin 2), Opa1 (optric atrophy 1), Tomm40 (translocase of outermembrane 40) and CypD (cyclophilin D) in grade III and grade IV HD patients and controls. The mutant Htt oligomers and the mitochondrial structural proteins were quantified in the striatum and frontal cortex of HD patients. Changes in expressions of the electron transport chain genes were found in HD patients and may represent a compensatory response to mitochondrial damage caused by mutant Htt. Increased expression of Drp1 and Fis1 and decreased expression of Mfn1, Mfn2, Opa1 and Tomm40 were found in HD patients relative to the controls. CypD was upregulated in HD patients, and this upregulation increased as HD progressed. Significantly increased immunoreactivity of 8-hydroxy-guanosine was found in the cortical specimens from stage III and IV HD patients relative to controls, suggesting increased oxidative DNA damage in HD patients. In contrast, significantly decreased immunoreactivities of cytochrome oxidase 1 and cytochrome b were found in HD patients relative to controls, indicating a loss of mitochondrial function in HD patients. Immunoblotting analysis revealed 15, 25 and 50 kDa mutant Htt oligomers in the brain specimens of HD patients. All oligomeric forms of mutant Htt were significantly increased in the cortical tissues of HD patients, and mutant Htt oligomers were found in the nucleus and in mitochondria. The increase in Drp1, Fis1 and CypD and the decrease in Mfn1 and Mfn2 may be responsible for abnormal mitochondrial dynamics that we found in the cortex of HD patients, and may contribute to neuronal damage in HD patients. The presence of mutant Htt

  19. Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons

    Directory of Open Access Journals (Sweden)

    McMurray Cynthia T

    2009-07-01

    Full Text Available Abstract Background Huntington's disease (HD is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP compounds efficiently inhibited formation of amyloid-β (Aβ aggregates in cell and mouse models representing Alzheimer's Disease (AD. In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD. Results TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons. Conclusion We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD.

  20. A SNP in the HTT promoter alters NF-κB binding and is a bidirectional genetic modifier of Huntington disease

    DEFF Research Database (Denmark)

    Bečanović, Kristina; Nørremølle, Anne; Neal, Scott J

    2015-01-01

    . HTT promoter analysis identified a NF-κB binding site that regulates HTT promoter transcriptional activity. A non-coding SNP, rs13102260:G > A, in this binding site impaired NF-κB binding and reduced HTT transcriptional activity and HTT protein expression. The presence of the rs13102260 minor (A...

  1. Cytodifferentiation of the postnatal mouse stomach in normal and Huntingtin-interacting protein 1-related-deficient mice

    Science.gov (United States)

    Keeley, Theresa M.

    2010-01-01

    Huntingtin-interacting protein 1-related (Hip1r) is highly expressed in gastric parietal cells, where it participates in vesicular trafficking associated with acid secretion. Hip1r-deficient mice have a progressive remodeling of the mucosa, including apoptotic loss of parietal cells, glandular hypertrophy, mucous cell metaplasia, and reduced numbers of zymogenic cells. In this study, we characterized gastric gland development in wild-type and Hip1r-deficient mice to define normal development, as well as the timing and sequence of the cellular transformation events in the mutant stomach. Postnatal (newborn to 8-wk-old) stomachs were examined by histological and gene expression analysis. At birth, gastric glands in wild-type and mutant mice were rudimentary and mature gastric epithelial cells were not apparent, although marker expression was detected for most cell lineages. Interestingly, newborns exhibited unusual cell types, including a novel surface cell filled with lipid and cells that coexpressed markers of mature mucous neck and zymogenic cells. Glandular morphogenesis proceeded rapidly in both genotypes, with gastric glands formed by weaning at 3 wk of age. In the Hip1r-deficient stomach, epithelial cell remodeling developed in a progressive manner. Initially, in the perinatal stomach, cellular changes were limited to parietal cell apoptosis. Other epithelial cell changes, including apoptotic loss of zymogenic cells and expansion of metaplastic mucous cells, emerged several weeks later when the glands were morphologically mature. Thus, parietal cell loss appeared to be the initiating event in Hip1r-deficient mice, with secondary remodeling of the other gastric epithelial cells. PMID:20813912

  2. Cloning, expression, purification, crystallization and preliminary crystallographic analysis of pseudo death-effector domain of HIPPI, a molecular partner of Huntingtin-interacting protein HIP-1

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Manisha; Majumder, Pritha; Bhattacharyya, Nitai P.; Dattagupta, Jiban K.; Sen, Udayaditya, E-mail: udayaditya.sen@saha.ac.in [Structural Genomics Section, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700064 (India)

    2006-12-01

    A pseudo death-effector domain (pDED) of HIPPI, a partner of Huntingtin-interacting protein HIP1, has been cloned, overexpressed and crystallized. The crystals of pDED-HIPPI diffracted to 2.2 Å. The formation of a heterodimer between Huntingtin-interacting protein-1 (HIP-1) and its novel partner HIPPI (HIP-1 protein interactor) through their pseudo death-effector domains (pDEDs) is a key step that recruits caspase-8 and initiates apoptosis. This could be one of the pathways by which apoptosis is increased in Huntington’s disease (HD). A construct consisting of the pDED of HIPPI has been cloned and overexpressed as 6NH-tagged protein and purified by Ni–NTA affinity chromatography. Crystals of the pDED of HIPPI were grown in space group P4{sub 1}, with unit-cell parameters a = b = 77.42, c = 33.31 Å and a calculated Matthews coefficient of 1.88 Å{sup 3} Da{sup −1} (33% solvent content) with two molecules per asymmetric unit.

  3. Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin

    Directory of Open Access Journals (Sweden)

    Hannah Girstmair

    2013-01-01

    Full Text Available Huntington disease (HD, a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ repeat in huntingtin (Htt, displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNAGln-CUG that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNAGln-CUG. The concentration of tRNAGln-CUG also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels.

  4. Depletion of cognate charged transfer RNA causes translational frameshifting within the expanded CAG stretch in huntingtin.

    Science.gov (United States)

    Girstmair, Hannah; Saffert, Paul; Rode, Sascha; Czech, Andreas; Holland, Gudrun; Bannert, Norbert; Ignatova, Zoya

    2013-01-31

    Huntington disease (HD), a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ) repeat in huntingtin (Htt), displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNA)(Gln-CUG) that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNA(Gln-CUG). The concentration of tRNA(Gln-CUG) also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels.

  5. Structural formation of huntingtin-like aggregates probed by small-angle neutron scattering

    Energy Technology Data Exchange (ETDEWEB)

    Stanley, Christopher B [ORNL; Perevozchikova, Tatiana [ORNL; Berthelier-Jung, Valerie M [ORNL

    2011-01-01

    In several neurodegenerative disorders, including Huntington s disease (HD), aspects concerning the earliest of protein structures that form along the aggregation pathway have increasingly gained attention since these particular species are likely to be neurotoxic. We used time-resolved small-angle neutron scattering (SANS) to probe in solution these transient structures formed by peptides having the N-terminal sequence context of mutant huntingtin (Htt) exon 1. We obtained snapshots of the formed aggregates as the kinetic reaction ensued to yield quantitative information on their size and mass. At the early stage, small precursor species with an initial radius of gyration (Rg) of 16.1 5.9 and average mass of a dimer to trimer were monitored. Structural growth was treated as two modes with a transition from three-dimensional early aggregate formation to two-dimensional fibril growth and association. Our SANS results on the internal structure of the mature fibrils demonstrate loose packing with about 1 peptide per 4.75 -sheet repeat distance, which is shown to be quantitatively consistent with a -helix model. This research provides new insights into the structures forming along the pathway of Htt exon 1 aggregation and should assist in determining the role that precursors play in neuronal toxicity.

  6. HTT-lowering reverses Huntington’s disease immune dysfunction caused by NFκB pathway dysregulation

    Science.gov (United States)

    Träger, Ulrike; Andre, Ralph; Lahiri, Nayana; Magnusson-Lind, Anna; Weiss, Andreas; Grueninger, Stephan; McKinnon, Chris; Sirinathsinghji, Eva; Kahlon, Shira; Pfister, Edith L.; Moser, Roger; Hummerich, Holger; Antoniou, Michael; Bates, Gillian P.; Luthi-Carter, Ruth; Lowdell, Mark W.; Björkqvist, Maria; Ostroff, Gary R.; Aronin, Neil

    2014-01-01

    Huntington’s disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington’s disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington’s disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function–the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington’s disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington’s disease. PMID:24459107

  7. Huntingtin-interacting Proteins, HIP14 and HIP14L, Mediate Dual Functions, Palmitoyl Acyltransferase and Mg2+ Transport*S⃞

    Science.gov (United States)

    Goytain, Angela; Hines, Rochelle M.; Quamme, Gary A.

    2008-01-01

    Polyglutamine expansions of huntingtin protein are responsible for the Huntington neurological disorder. HIP14 protein has been shown to interact with huntingtin. HIP14 and a HIP14-like protein, HIP14L, with a 69% similarity reside in the Golgi and possess palmitoyl acyltransferase activity through innate cysteine-rich domains, DHHC. Here, we used microarray analysis to show that reduced extracellular magnesium concentration increases HIP14L mRNA suggesting a role in cellular magnesium metabolism. Because HIP14 was not on the microarray platform, we used real-time reverse transcriptase-PCR to show that HIP14 and HIP14L transcripts were up-regulated 3-fold with low magnesium. Western analysis with a specific HIP14 antibody also showed that endogenous HIP14 protein increased with diminished magnesium. Furthermore, we demonstrate that when expressed in Xenopus oocytes, HIP14 and HIP14L mediate Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with Michaelis constants of 0.87 ± 0.02 and 0.74 ± 0.07 mm, respectively. Diminished magnesium leads to an apparent increase in HIP14-green fluorescent protein and HIP14L-green fluorescent fusion proteins in the Golgi complex and subplasma membrane post-Golgi vesicles of transfected epithelial cells. We also show that inhibition of palmitoylation with 2-bromopalmitate, or deletion of the DHHC motif HIP14ΔDHHC, diminishes HIP14-mediated Mg2+ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14ΔDHHC mutant restored Mg2+ transport to values observed with wild-type HIP14. Although we did not directly measure palmitoylation of HIP14 in these studies, the data are consistent with a regulatory role of autopalmitoylation in HIP14-mediated Mg2+ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg2+ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of

  8. Golgi Protein ACBD3 Mediates Neurotoxicity Associated with Huntington’s Disease

    Directory of Open Access Journals (Sweden)

    Juan I. Sbodio

    2013-09-01

    Full Text Available Huntington’s disease (HD is an autosomal-dominant neurodegenerative disease caused by the expansion of polyglutamine repeats in the gene for huntingtin (Htt. In HD, the corpus striatum selectively degenerates despite the uniform expression of mutant huntingtin (mHtt throughout the brain and body. Striatal selectivity reflects the binding of the striatal-selective protein Rhes to mHtt to augment cytotoxicity, but molecular mechanisms underlying the toxicity have been elusive. Here, we report that the Golgi protein acyl-CoA binding domain containing 3 (ACBD3 mediates mHtt cytotoxicity via a Rhes/mHtt/ACBD3 complex. ACBD3 levels are markedly elevated in the striatum of HD patients, in a striatal cell line harboring polyglutamine repeats, and in the brains of HD mice. Moreover, ACBD3 deletion abolishes HD neurotoxicity, which is increased by ACBD3 overexpression. Enhanced levels of ACBD3 elicited by endoplasmic reticulum, mitochondrial, and Golgi stresses may account for HD-associated augmentation of ACBD3 and neurodegeneration.

  9. Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington's Disease.

    Science.gov (United States)

    Miniarikova, Jana; Zanella, Ilaria; Huseinovic, Angelina; van der Zon, Tom; Hanemaaijer, Evelyn; Martier, Raygene; Koornneef, Annemart; Southwell, Amber L; Hayden, Michael R; van Deventer, Sander J; Petry, Harald; Konstantinova, Pavlina

    2016-03-22

    Huntington's disease (HD) is a neurodegenerative disorder caused by accumulation of CAG expansions in the huntingtin (HTT) gene. Hence, decreasing the expression of mutated HTT (mtHTT) is the most upstream approach for treatment of HD. We have developed HTT gene-silencing approaches based on expression cassette-optimized artificial miRNAs (miHTTs). In the first approach, total silencing of wild-type and mtHTT was achieved by targeting exon 1. In the second approach, allele-specific silencing was induced by targeting the heterozygous single-nucleotide polymorphism (SNP) rs362331 in exon 50 or rs362307 in exon 67 linked to mtHTT. The miHTT expression cassette was optimized by embedding anti-HTT target sequences in ten pri-miRNA scaffolds and their HTT knockdown efficacy, allele selectivity, passenger strand activity, and processing patterns were analyzed in vitro. Furthermore, three scaffolds expressing miH12 targeting exon 1 were incorporated in an adeno-associated viral serotype 5 (AAV5) vector and their HTT knock-down efficiency and pre-miHTT processing were compared in the humanized transgenic Hu128/21 HD mouse model. Our data demonstrate strong allele-selective silencing of mtHTT by miSNP50 targeting rs362331 and total HTT silencing by miH12 both in vitro and in vivo. Ultimately, we show that HTT knock-down efficiency and guide strand processing can be enhanced by using different cellular pri-miRNA scaffolds.

  10. Full-length huntingtin levels modulate body weight by influencing insulin-like growth factor 1 expression

    DEFF Research Database (Denmark)

    Pouladi, Mahmoud A; Xie, Yuanyun; Skotte, Niels Henning

    2010-01-01

    body weight by modulating the IGF-1 pathway. Plasma IGF-1 levels correlate with body weight and htt levels in the transgenic YAC mice expressing human htt. The effect of htt on IGF-1 expression is independent of CAG size. No effect on body weight is observed in transgenic YAC mice expressing...... and decreases the body weight of YAC128 animals to WT levels. Furthermore, given the ubiquitous expression of IGF-1 within the central nervous system, we also examined the impact of FL htt levels on IGF-1 expression in different regions of the brain, including the striatum, cerebellum of YAC18, YAC128......Levels of full-length huntingtin (FL htt) influence organ and body weight, independent of polyglutamine length. The growth hormone-insulin like growth factor-1 (GH-IGF-1) axis is well established as a regulator of organ growth and body weight. In this study, we investigate the involvement...

  11. Effects of Intracellular Expression of Anti-Huntingtin Antibodies of Various Specificities on Mutant Huntingtin Aggregation and Toxicity

    National Research Council Canada - National Science Library

    Ali Khoshnan; Jan Ko; Paul H. Patterson

    2002-01-01

    ... (htt) protein. In the brains of Huntington's disease (HD) mouse models, the anti-polyQ mAbs bind to various cytoplasmic compartments, whereas the anti-polyP and anti-C terminus mAbs bind nuclear inclusions containing htt...

  12. Detection of ubiquitinated huntingtin species in intracellular aggregates

    Directory of Open Access Journals (Sweden)

    Katrin eJuenemann

    2015-01-01

    Full Text Available Protein conformation diseases, including polyglutamine diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease is one of nine diseases caused by an expanded polyglutamine repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated huntingtin such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated huntingtin fragments offers an important possibility to understand huntingtin degradation and aggregation processes within the cell. For the identification of aggregated huntingtin and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant huntingtin. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

  13. Aggregation of scaffolding protein DISC1 dysregulates phosphodiesterase 4 in Huntington's disease.

    Science.gov (United States)

    Tanaka, Motomasa; Ishizuka, Koko; Nekooki-Machida, Yoko; Endo, Ryo; Takashima, Noriko; Sasaki, Hideyuki; Komi, Yusuke; Gathercole, Amy; Huston, Elaine; Ishii, Kazuhiro; Hui, Kelvin Kai-Wan; Kurosawa, Masaru; Kim, Sun-Hong; Nukina, Nobuyuki; Takimoto, Eiki; Houslay, Miles D; Sawa, Akira

    2017-04-03

    Huntington's disease (HD) is a polyglutamine (polyQ) disease caused by aberrant expansion of the polyQ tract in Huntingtin (HTT). While motor impairment mediated by polyQ-expanded HTT has been intensively studied, molecular mechanisms for nonmotor symptoms in HD, such as psychiatric manifestations, remain elusive. Here we have demonstrated that HTT forms a ternary protein complex with the scaffolding protein DISC1 and cAMP-degrading phosphodiesterase 4 (PDE4) to regulate PDE4 activity. We observed pathological cross-seeding between DISC1 and mutant HTT aggregates in the brains of HD patients as well as in a murine model that recapitulates the polyQ pathology of HD (R6/2 mice). In R6/2 mice, consequent reductions in soluble DISC1 led to dysregulation of DISC1-PDE4 complexes, aberrantly increasing the activity of PDE4. Importantly, exogenous expression of a modified DISC1, which binds to PDE4 but not mutant HTT, normalized PDE4 activity and ameliorated anhedonia in the R6/2 mice. We propose that cross-seeding of mutant HTT and DISC1 and the resultant changes in PDE4 activity may underlie the pathology of a specific subset of mental manifestations of HD, which may provide an insight into molecular signaling in mental illness in general.

  14. CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

    Directory of Open Access Journals (Sweden)

    Ashley A. Zurawel

    2016-09-01

    Full Text Available Proteins containing polyglutamine (polyQ regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD. To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms.

  15. High Protein Diet and Huntington's Disease.

    Directory of Open Access Journals (Sweden)

    Chiung-Mei Chen

    Full Text Available Huntington's disease (HD is a neurodegenerative disorder caused by the huntingtin (HTT gene with expanded CAG repeats. In addition to the apparent brain abnormalities, impairments also occur in peripheral tissues. We previously reported that mutant Huntingtin (mHTT exists in the liver and causes urea cycle deficiency. A low protein diet (17% restores urea cycle activity and ameliorates symptoms in HD model mice. It remains unknown whether the dietary protein content should be monitored closely in HD patients because the normal protein consumption is lower in humans (~15% of total calories than in mice (~22%. We assessed whether dietary protein content affects the urea cycle in HD patients. Thirty HD patients were hospitalized and received a standard protein diet (13.7% protein for 5 days, followed by a high protein diet (HPD, 26.3% protein for another 5 days. Urea cycle deficiency was monitored by the blood levels of citrulline and ammonia. HD progression was determined by the Unified Huntington's Disease Rating Scale (UHDRS. The HPD increased blood citrulline concentration from 15.19 μmol/l to 16.30 μmol/l (p = 0.0378 in HD patients but did not change blood ammonia concentration. A 2-year pilot study of 14 HD patients found no significant correlation between blood citrulline concentration and HD progression. Our results indicated a short period of the HPD did not markedly compromise urea cycle function. Blood citrulline concentration is not a reliable biomarker of HD progression.

  16. Glial cell lineage expression of mutant ataxin-1 and huntingtin induces developmental and late-onset neuronal pathologies in Drosophila models.

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    Takuya Tamura

    Full Text Available BACKGROUND: In several neurodegenerative disorders, toxic effects of glial cells on neurons are implicated. However the generality of the non-cell autonomous pathologies derived from glial cells has not been established, and the specificity among different neurodegenerative disorders remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We newly generated Drosophila models expressing human mutant huntingtin (hHtt103Q or ataxin-1 (hAtx1-82Q in the glial cell lineage at different stages of differentiation, and analyzed their morphological and behavioral phenotypes. To express hHtt103Q and hAtx1-82Q, we used 2 different Gal4 drivers, gcm-Gal4 and repo-Gal4. Gcm-Gal4 is known to be a neuroglioblast/glioblast-specific driver whose effect is limited to development. Repo-Gal4 is known to be a pan-glial driver and the expression starts at glioblasts and continues after terminal differentiation. Gcm-Gal4-induced hHtt103Q was more toxic than repo-Gal4-induced hHtt103Q from the aspects of development, locomotive activity and survival of flies. When hAtx1-82Q was expressed by gcm- or repo-Gal4 driver, no fly became adult. Interestingly, the head and brain sizes were markedly reduced in a part of pupae expressing hAtx1-82Q under the control of gcm-Gal4, and these pupae showed extreme destruction of the brain structure. The other pupae expressing hAtx1-82Q also showed brain shrinkage and abnormal connections of neurons. These results suggested that expression of polyQ proteins in neuroglioblasts provided a remarkable effect on the developmental and adult brains, and that glial cell lineage expression of hAtx1-82Q was more toxic than that of hHtt103Q in our assays. CONCLUSION/SIGNIFICANCE: All these studies suggested that the non-cell autonomous effect of glial cells might be a common pathology shared by multiple neurodegenerative disorders. In addition, the fly models would be available for analyzing molecular pathologies and developing novel therapeutics against

  17. Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    Directory of Open Access Journals (Sweden)

    Laureen Jacquet

    2016-03-01

    Full Text Available The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  18. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.

    2003-01-01

    or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...... of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. Methods NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...

  19. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.;

    2003-01-01

    of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. Methods NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...... or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...

  20. Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach.

    Science.gov (United States)

    Broncel, Malgorzata; Serwa, Remigiusz A; Bunney, Tom D; Katan, Matilda; Tate, Edward W

    2016-02-01

    AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE substrates have been identified by immunoprecipitation and mass spectrometry approaches, and the full range of targets is yet to be determined in mammalian cells. We describe here the first example of global chemoproteomic screening and substrate validation for HYPE-mediated AMPylation in mammalian cell lysate. Through quantitative mass-spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification, we identified a total of 25 AMPylated proteins, including the previously validated substrate endoplasmic reticulum (ER) chaperone BiP (HSPA5), and also novel substrates involved in pathways of gene expression, ATP biosynthesis, and maintenance of the cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.

  1. Selective expression of mutant huntingtin during development recapitulates characteristic features of Huntington's disease.

    Science.gov (United States)

    Molero, Aldrin E; Arteaga-Bracho, Eduardo E; Chen, Christopher H; Gulinello, Maria; Winchester, Michael L; Pichamoorthy, Nandini; Gokhan, Solen; Khodakhah, Kamran; Mehler, Mark F

    2016-05-17

    Recent studies have identified impairments in neural induction and in striatal and cortical neurogenesis in Huntington's disease (HD) knock-in mouse models and associated embryonic stem cell lines. However, the potential role of these developmental alterations for HD pathogenesis and progression is currently unknown. To address this issue, we used BACHD:CAG-Cre(ERT2) mice, which carry mutant huntingtin (mHtt) modified to harbor a floxed exon 1 containing the pathogenic polyglutamine expansion (Q97). Upon tamoxifen administration at postnatal day 21, the floxed mHtt-exon1 was removed and mHtt expression was terminated (Q97(CRE)). These conditional mice displayed similar profiles of impairments to those mice expressing mHtt throughout life: (i) striatal neurodegeneration, (ii) early vulnerability to NMDA-mediated excitotoxicity, (iii) impairments in motor coordination, (iv) temporally distinct abnormalities in striatal electrophysiological activity, and (v) altered corticostriatal functional connectivity and plasticity. These findings strongly suggest that developmental aberrations may play important roles in HD pathogenesis and progression.

  2. Clues to γ-secretase, huntingtin and Hirano body normal function using the model organism Dictyostelium discoideum

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    Myre Michael A

    2012-04-01

    Full Text Available Abstract Many neurodegenerative disorders, although related by their destruction of brain function, display remarkable cellular and/or regional pathogenic specificity likely due to a deregulated functionality of the mutant protein. However, neurodegenerative disease genes, for example huntingtin (HTT, the ataxins, the presenilins (PSEN1/PSEN2 are not simply localized to neurons but are ubiquitously expressed throughout peripheral tissues; it is therefore paramount to properly understand the earliest precipitating events leading to neuronal pathogenesis to develop effective long-term therapies. This means, in no unequivocal terms, it is crucial to understand the gene's normal function. Unfortunately, many genes are often essential for embryogenesis which precludes their study in whole organisms. This is true for HTT, the β-amyloid precursor protein (APP and presenilins, responsible for early onset Alzheimer's disease (AD. To better understand neurological disease in humans, many lower and higher eukaryotic models have been established. So the question arises: how reasonable is the use of organisms to study neurological disorders when the model of choice does not contain neurons? Here we will review the surprising, and novel emerging use of the model organism Dictyostelium discoideum, a species of soil-living amoeba, as a valuable biomedical tool to study the normal function of neurodegenerative genes. Historically, the evidence on the usefulness of simple organisms to understand the etiology of cellular pathology cannot be denied. But using an organism without a central nervous system to understand diseases of the brain? We will first introduce the life cycle of Dictyostelium, the presence of many disease genes in the genome and how it has provided unique opportunities to identify mechanisms of disease involving actin pathologies, mitochondrial disease, human lysosomal and trafficking disorders and host-pathogen interactions. Secondly, I will

  3. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

    OpenAIRE

    Hoop, Cody L.; Lin, Hsiang-Kai; Kar, Karunakar; Magyarfalvi, Gábor; Lamley, Jonathan M.; Boatz, Jennifer C.; Mandal, Abhishek; Lewandowski, Józef R.; Wetzel, Ronald; van der Wel, Patrick C.A.

    2016-01-01

    Huntington’s disease is a devastating and incurable inherited neurodegenerative disease. Like at least eight other diseases, its primary genetic cause is the CAG repeat expansion in a specific gene. Mutant huntingtin protein undergoes misfolding and aggregation, causing degeneration of neurons through as-yet poorly understood mechanisms. Attempts to characterize the implicated protein deposits have until now had limited success. We present our structural studies of mutant huntingtin-derived p...

  4. PACSIN 1 interacts with huntingtin and is absent from synaptic varicosities in presymptomatic Huntington's disease brains.

    Science.gov (United States)

    Modregger, Jan; DiProspero, Nicholas A; Charles, Vinod; Tagle, Danilo A; Plomann, Markus

    2002-10-01

    Huntington's disease (HD) is caused by a pathological expansion of a CAG repeat in the first exon of the gene coding for huntingtin, resulting in an abnormally long polyglutamine stretch. Despite its widespread expression, mutant huntingtin leads to selective neuronal loss in the striatum and cortex. Here we report that the neurospecific phosphoprotein PACSIN 1, which has been implicated as playing a central role in synaptic vesicle recycling, interacts with huntingtin via its C-terminal SH3 domain. Moreover, two other isoforms, PACSIN 2 and 3, which show a wider tissue distribution including the brain, do not interact with huntingtin despite a highly conserved SH3 domain. Furthermore, this interaction is repeat-length-dependent and is enhanced with mutant huntingtin, possibly causing the sequestration of PACSIN 1. Normally, PACSIN 1 is located along neurites and within synaptic boutons, but in HD patient neurons, there is a progressive loss of PACSIN 1 immunostaining in synaptic varicosities, beginning in presymptomatic and early-stage HD. Further, PACSIN 1 immunostaining of HD patient tissue reveals a more cytoplasmic distribution of the protein, with particular concentration in the perinuclear region coincident with mutant huntingtin. Thus, the specific interaction of huntingtin with the neuronal PACSIN isoform, PACSIN 1, and its altered intracellular distribution in pathological tissue, together with the observed differences in the binding behavior, suggest a role for PACSIN 1 during early stages of the selective neuropathology of HD.

  5. Recent Trends in Detection of Huntingtin and Preclinical Models of Huntington’s Disease

    OpenAIRE

    2014-01-01

    Huntington’s disease is a genetically inherited neurodegenerative disease that is characterized by neuronal cell death in the brain. Molecular biology techniques to detect and quantify huntingtin protein in biological samples involve fluorescence imaging, western blotting, and PCR. Modified cell lines are widely used as models for Huntington’s disease for preclinical screening of drugs to study their ability to suppress the expression of huntingtin. Although worm and fly species have been exp...

  6. HD CAGnome: a search tool for huntingtin CAG repeat length-correlated genes.

    Directory of Open Access Journals (Sweden)

    Ekaterina I Galkina

    Full Text Available BACKGROUND: The length of the huntingtin (HTT CAG repeat is strongly correlated with both age at onset of Huntington's disease (HD symptoms and age at death of HD patients. Dichotomous analysis comparing HD to controls is widely used to study the effects of HTT CAG repeat expansion. However, a potentially more powerful approach is a continuous analysis strategy that takes advantage of all of the different CAG lengths, to capture effects that are expected to be critical to HD pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: We used continuous and dichotomous approaches to analyze microarray gene expression data from 107 human control and HD lymphoblastoid cell lines. Of all probes found to be significant in a continuous analysis by CAG length, only 21.4% were so identified by a dichotomous comparison of HD versus controls. Moreover, of probes significant by dichotomous analysis, only 33.2% were also significant in the continuous analysis. Simulations revealed that the dichotomous approach would require substantially more than 107 samples to either detect 80% of the CAG-length correlated changes revealed by continuous analysis or to reduce the rate of significant differences that are not CAG length-correlated to 20% (n = 133 or n = 206, respectively. Given the superior power of the continuous approach, we calculated the correlation structure between HTT CAG repeat lengths and gene expression levels and created a freely available searchable website, "HD CAGnome," that allows users to examine continuous relationships between HTT CAG and expression levels of ∼20,000 human genes. CONCLUSIONS/SIGNIFICANCE: Our results reveal limitations of dichotomous approaches compared to the power of continuous analysis to study a disease where human genotype-phenotype relationships strongly support a role for a continuum of CAG length-dependent changes. The compendium of HTT CAG length-gene expression level relationships found at the HD CAGnome now provides

  7. Huntingtin-lowering strategies in Huntington's disease: antisense oligonucleotides, small RNAs, and gene editing.

    Science.gov (United States)

    Aronin, Neil; DiFiglia, Marian

    2014-09-15

    The idea to lower mutant huntingtin is especially appealing in Huntington's disease (HD). It is autosomal dominant, so that expression of the mutant allele causes the disease. Advances in RNA and gene regulation provide foundations for the huntingtin gene (both normal and mutant alleles) and possibly the mutant allele only. There is much preclinical animal work to support the concept of gene and RNA silencing, but, to date, no clinical studies have been attempted in HD. Preventing expression of mutant huntingtin protein is at the cusp for a human trial. Antisense oligonucleotides delivered to patients with amyotrophic lateral sclerosis have been well tolerated; small RNAs administered to rodent and nonhuman primate brain knocked down huntingtin messenger RNA (mRNA); short-hairpin complementary DNA of microRNAs can be expressed in adeno-associated virus to provide long-term silencing of huntingtin mRNA and protein. We expect that these approaches will be ready for clinical studies in the near future, once safety has been validated. Our understanding of gene editing-changing the huntingtin gene itself-is rapidly progressing. Harnessing our knowledge of transcription and translation should push scientific creativity to new and exciting advances that overcome the lethality of the mutant gene in HD. © 2014 International Parkinson and Movement Disorder Society.

  8. Essential genetic interactors of SIR2 required for spatial sequestration and asymmetrical inheritance of protein aggregates.

    Directory of Open Access Journals (Sweden)

    Jia Song

    2014-07-01

    Full Text Available Sir2 is a central regulator of yeast aging and its deficiency increases daughter cell inheritance of stress- and aging-induced misfolded proteins deposited in aggregates and inclusion bodies. Here, by quantifying traits predicted to affect aggregate inheritance in a passive manner, we found that a passive diffusion model cannot explain Sir2-dependent failures in mother-biased segregation of either the small aggregates formed by the misfolded Huntingtin, Htt103Q, disease protein or heat-induced Hsp104-associated aggregates. Instead, we found that the genetic interaction network of SIR2 comprises specific essential genes required for mother-biased segregation including those encoding components of the actin cytoskeleton, the actin-associated myosin V motor protein Myo2, and the actin organization protein calmodulin, Cmd1. Co-staining with Hsp104-GFP demonstrated that misfolded Htt103Q is sequestered into small aggregates, akin to stress foci formed upon heat stress, that fail to coalesce into inclusion bodies. Importantly, these Htt103Q foci, as well as the ATPase-defective Hsp104Y662A-associated structures previously shown to be stable stress foci, co-localized with Cmd1 and Myo2-enriched structures and super-resolution 3-D microscopy demonstrated that they are associated with actin cables. Moreover, we found that Hsp42 is required for formation of heat-induced Hsp104Y662A foci but not Htt103Q foci suggesting that the routes employed for foci formation are not identical. In addition to genes involved in actin-dependent processes, SIR2-interactors required for asymmetrical inheritance of Htt103Q and heat-induced aggregates encode essential sec genes involved in ER-to-Golgi trafficking/ER homeostasis.

  9. The HTT CAG-Expansion Mutation Determines Age at Death but Not Disease Duration in Huntington Disease.

    Science.gov (United States)

    Keum, Jae Whan; Shin, Aram; Gillis, Tammy; Mysore, Jayalakshmi Srinidhi; Abu Elneel, Kawther; Lucente, Diane; Hadzi, Tiffany; Holmans, Peter; Jones, Lesley; Orth, Michael; Kwak, Seung; MacDonald, Marcy E; Gusella, James F; Lee, Jong-Min

    2016-02-04

    Huntington disease (HD) is caused by an expanded HTT CAG repeat that leads in a length-dependent, completely dominant manner to onset of a characteristic movement disorder. HD also displays early mortality, so we tested whether the expanded CAG repeat exerts a dominant influence on age at death and on the duration of clinical disease. We found that, as with clinical onset, HD age at death is determined by expanded CAG-repeat length and has no contribution from the normal CAG allele. Surprisingly, disease duration is independent of the mutation's length. It is also unaffected by a strong genetic modifier of HD motor onset. These findings suggest two parsimonious alternatives. (1) HD pathogenesis is driven by mutant huntingtin, but before or near motor onset, sufficient CAG-driven damage occurs to permit CAG-independent processes and then lead to eventual death. In this scenario, some pathological changes and their clinical correlates could still worsen in a CAG-driven manner after disease onset, but these CAG-related progressive changes do not themselves determine duration. Alternatively, (2) HD pathogenesis is driven by mutant huntingtin acting in a CAG-dependent manner with different time courses in multiple cell types, and the cellular targets that lead to motor onset and death are different and independent. In this scenario, processes driven by HTT CAG length lead directly to death but not via the striatal pathology associated with motor manifestations. Each scenario has important ramifications for the design and testing of potential therapeutics, especially those aimed at preventing or delaying characteristic motor manifestations.

  10. Protein aggregates in Huntington’s disease

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    Arrasate, Montserrat; Finkbeiner, Steven

    2014-01-01

    Huntington’s disease (HD) is an incurable neurodegenerative disease characterized by abnormal motor movements, personality changes, and early death. HD is caused by a mutation in the IT-15 gene that expands abnormally the number of CAG nucleotide repeats. As a result, the translated protein huntingtin contains disease-causing expansions of glutamines (polyQ) that make it prone to misfold and aggregate. While the gene and mutations that cause HD are known, the mechanisms underlying HD pathogenesis are not. Here we will review the state of knowledge of HD, focusing especially on a hallmark pathological feature—intracellular aggregates of mutant Htt called inclusion bodies (IBs). We will describe the role of IBs in the disease. We speculate that IB formation could be just one component of a broader coping response triggered by misfolded Htt whose efficacy may depend on the extent to which it clears toxic forms of mutant Htt. We will describe how IB formation might be regulated and which factors could determine different coping responses in different subsets of neurons. A differential regulation of IB formation as a function of the cellular context could, eventually, explain part of the neuronal vulnerability observed in HD. PMID:22200539

  11. TRiC’s tricks inhibit huntingtin aggregation

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    Shahmoradian, Sarah H; Galaz-Montoya, Jesus G; Schmid, Michael F; Cong, Yao; Ma, Boxue; Spiess, Christoph; Frydman, Judith; Ludtke, Steven J; Chiu, Wah

    2013-01-01

    In Huntington’s disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC’s inhibition of mhttQ51 aggregation in vitro. DOI: http://dx.doi.org/10.7554/eLife.00710.001 PMID:23853712

  12. Serotonin transporter gene (5-HTT) polymorphisms and compulsive buying.

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    Devor, E J; Magee, H J; Dill-Devor, R M; Gabel, J; Black, D W

    1999-04-16

    We examined a panel of 21 patients diagnosed with compulsive buying for two DNA sequence polymorphisms found in the gene that encodes the serotonin transport (5-HTT). One polymorphism, found in the promoter region of the 5-HTT gene, involves a 44-base pair (bp) deletion, and the other, found in the second intron, is due to variable numbers of a repeat sequence. We also typed a panel of 38 psychiatrically normal controls for both 5-HH markers. When compared to this control panel, no significant differences were seen for either 5-HTT marker among the compulsive buyers.

  13. The Aggregation of Huntingtin and α-Synuclein

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    María Elena Chánez-Cárdenas

    2012-01-01

    Full Text Available Huntington’s and Parkinson’s diseases are neurodegenerative disorders associated with unusual protein interactions. Although the origin and evolution of these diseases are completely different, characteristic deposits of protein aggregates (huntingtin and α-synuclein resp., are a common feature in both diseases. After these observations, many studies are performed with both proteins. Some of them try to understand the nature and driving forces of the aggregation process; others try to find a correlation between the genetic and failure in protein function. Finally with the combination of both approaches, it was proposed that possible strategies deal with pathologic aggregation. Unfortunately, if protein aggregation is a cause or a consequence of the neurodegeneration observed in these pathologies, it is still debatable. This paper describes the process of aggregation of two proteins: huntingtin and α synuclein. The characteristics of the aggregation reaction of these proteins have been followed with novel methods both in vivo and in vitro; these studies include both the combination with other proteins and the presence of various chemical compounds. The ultimate goal of this study was to summarize recent findings on protein aggregation and its possible role as a therapeutic target in neurodegenerative diseases and their role in biomaterial science.

  14. Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

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    Julia F Alterman

    2015-01-01

    Full Text Available Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

  15. Subcellular clearance and accumulation of Huntington disease protein: A mini-review

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    Ting eZhao

    2016-04-01

    Full Text Available Huntington’s disease (HD is an autosomal dominant, progressive neurodegenerative disease caused by an expanded polyglutamine (polyQ tract in the N-terminal region of mutant huntingtin (mHtt. As a result, mHtt forms aggregates that are abundant in the nuclei and processes of neuronal cells. Although the roles of mHtt aggregates are still debated, the formation of aggregates points to deficient clearance of mHtt in brain cells. Since the accumulation of mHtt is a prerequisite for its neurotoxicity, exploring the mechanisms for mHtt accumulation and clearance would advance our understanding of HD pathogenesis and help us develop treatments for HD. We know that the ubiquitin-proteasome system and autophagy play important roles in clearing mHtt; however, how mHtt preferentially accumulates in neuronal nuclei and processes remains unclear. Studying the clearance of mHtt in neuronal cells is a challenge because neurons are morphologically and functionally polarized, which means the turnover of mHtt may be distinct in different cellular compartments. In this review, we discuss our current knowledge about the clearance and accumulation of mHtt and strategies of examining mHtt clearance and accumulation in different subcellular regions

  16. Recent Trends in Detection of Huntingtin and Preclinical Models of Huntington's Disease.

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    Mantha, Neelima; Das, Nandita G; Das, Sudip K

    2014-01-01

    Huntington's disease is a genetically inherited neurodegenerative disease that is characterized by neuronal cell death in the brain. Molecular biology techniques to detect and quantify huntingtin protein in biological samples involve fluorescence imaging, western blotting, and PCR. Modified cell lines are widely used as models for Huntington's disease for preclinical screening of drugs to study their ability to suppress the expression of huntingtin. Although worm and fly species have been experimented on as models for Huntington's disease, the most successful animal models have been reported to be primates. This review critically analyses the molecular biology techniques for detection and quantitation of huntingtin and evaluates the various animal species for use as models for Huntington's disease.

  17. Rhes, a striatal-selective protein implicated in Huntington disease, binds beclin-1 and activates autophagy.

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    Mealer, Robert G; Murray, Alexandra J; Shahani, Neelam; Subramaniam, Srinivasa; Snyder, Solomon H

    2014-02-07

    The protein mutated in Huntington disease (HD), mutant huntingtin (mHtt), is expressed throughout the brain and body. However, the pathology of HD is characterized by early and dramatic destruction selectively of the striatum. We previously reported that the striatal-specific protein Rhes binds mHtt and enhances its cytotoxicity. Moreover, Rhes-deleted mice are dramatically protected from neurodegeneration and motor dysfunction in mouse models of HD. We now report a function of Rhes in autophagy, a lysosomal degradation pathway implicated in aging and HD neurodegeneration. In PC12 cells, deletion of endogenous Rhes decreases autophagy, whereas Rhes overexpression activates autophagy. These effects are independent of mTOR and opposite in the direction predicted by the known activation of mTOR by Rhes. Rhes robustly binds the autophagy regulator Beclin-1, decreasing its inhibitory interaction with Bcl-2 independent of JNK-1 signaling. Finally, co-expression of mHtt blocks Rhes-induced autophagy activation. Thus, the isolated pathology and delayed onset of HD may reflect the striatal-selective expression and changes in autophagic activity of Rhes.

  18. Mutant huntingtin gene-dose impacts on aggregate deposition, DARPP32 expression and neuroinflammation in HdhQ150 mice.

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    Douglas Young

    Full Text Available Huntington's disease (HD is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i an increase of neuronal intra-nuclear inclusions (NIIs in brain, (ii an increase of extra-nuclear aggregates in dentate gyrus, (iii a decrease of DARPP32 protein and (iv an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model.

  19. Expression of mutant huntingtin in leptin receptor-expressing neurons does not control the metabolic and psychiatric phenotype of the BACHD mouse.

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    Sofia Hult Lundh

    Full Text Available Metabolic and psychiatric disturbances occur early on in the clinical manifestation of Huntington's disease (HD, a neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT gene. Hypothalamus has emerged as an important site of pathology and alterations in this area and its neuroendocrine circuits may play a role in causing early non-motor symptoms and signs in HD. Leptin is a hormone that controls energy homeostasis by signaling through leptin receptors in the hypothalamus. Disturbed leptin action is implicated in both obesity and depression and altered circulating levels of leptin have been reported in both clinical HD and rodent models of the disease. Pathological leptin signaling may therefore be involved in causing the metabolic and psychiatric disturbances of HD. Here we tested the hypothesis that expression of mutant HTT in leptin receptor carrying neurons plays a role in the development of the non-motor phenotype in the BACHD mouse model. Our results show that inactivation of mutant HTT in leptin receptor-expressing neurons in the BACHD mouse using cross-breeding based on a cre-loxP system did not have an effect on the metabolic phenotype or anxiety-like behavior. The data suggest that mutant HTT disrupts critical hypothalamic pathways by other mechanisms than interfering with intracellular leptin signaling.

  20. Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors

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    Betschart Claudia

    2010-12-01

    Full Text Available Abstract Background The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB. CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown. Results Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec or selectively (LY-411,575 or DAPT reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin. Conclusion We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin

  1. HDAC4 reduction: a novel therapeutic strategy to target cytoplasmic huntingtin and ameliorate neurodegeneration.

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    Michal Mielcarek

    2013-11-01

    Full Text Available Histone deacetylase (HDAC 4 is a transcriptional repressor that contains a glutamine-rich domain. We hypothesised that it may be involved in the molecular pathogenesis of Huntington's disease (HD, a protein-folding neurodegenerative disorder caused by an aggregation-prone polyglutamine expansion in the huntingtin protein. We found that HDAC4 associates with huntingtin in a polyglutamine-length-dependent manner and co-localises with cytoplasmic inclusions. We show that HDAC4 reduction delayed cytoplasmic aggregate formation, restored Bdnf transcript levels, and rescued neuronal and cortico-striatal synaptic function in HD mouse models. This was accompanied by an improvement in motor coordination, neurological phenotypes, and increased lifespan. Surprisingly, HDAC4 reduction had no effect on global transcriptional dysfunction and did not modulate nuclear huntingtin aggregation. Our results define a crucial role for the cytoplasmic aggregation process in the molecular pathology of HD. HDAC4 reduction presents a novel strategy for targeting huntingtin aggregation, which may be amenable to small-molecule therapeutics.

  2. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core.

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    Hoop, Cody L; Lin, Hsiang-Kai; Kar, Karunakar; Magyarfalvi, Gábor; Lamley, Jonathan M; Boatz, Jennifer C; Mandal, Abhishek; Lewandowski, Józef R; Wetzel, Ronald; van der Wel, Patrick C A

    2016-02-09

    Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

  3. Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1 Are Essential for the Binding of Clathrin Light Chain

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    Joel A. Ybe

    2009-01-01

    Full Text Available The interaction between HIP family proteins (HIP1 and HIP12/1R and clathrin is fundamental to endocytosis. We used circular dichroism (CD to study the stability of an HIP1 subfragment (aa468-530 that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83C∘. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494. The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae, HIP1 from D. melanogaster, and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.

  4. Effect of 2'-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells.

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    Matsui, Masayuki; Threlfall, Richard N; Caruthers, Marvin H; Corey, David R

    2014-12-15

    Optimizing oligonucleotides as therapeutics will require exploring how chemistry can be used to enhance their effects inside cells. To achieve this goal it will be necessary to fully explore chemical space around the native DNA/RNA framework to define the potential of diverse chemical modifications. In this report we examine the potential of thiophosphonoacetate (thioPACE)-modified 2'-O-methyl oligoribonucleotides as inhibitors of human huntingtin (HTT) expression. Inhibition occurred, but was less than with analogous locked nucleic acid (LNA)-substituted oligomers lacking the thioPACE modification. These data suggest that thioPACE oligonucleotides have the potential to control gene expression inside cells. However, advantages relative to other modifications were not demonstrated. Additional modifications are likely to be necessary to fully explore any potential advantages of thioPACE substitutions.

  5. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

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    Tomoyuki Yamanaka

    Full Text Available In polyglutamine (polyQ diseases including Huntington's disease (HD, mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

  6. Non-cell autonomous cell death caused by transmission of Huntingtin aggregates in Drosophila.

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    Babcock, Daniel T; Ganetzky, Barry

    2015-01-01

    Recent evidence indicates that protein aggregates can spread between neurons in several neurodegenerative diseases but much remains unknown regarding the underlying mechanisms responsible for this spreading and its role in disease progression. We recently demonstrated that mutant Huntingtin aggregates spread between cells within the Drosophila brain resulting in non-cell autonomous loss of a pair of large neurons in the posterior protocerebrum. However, the full extent of neuronal loss throughout the brain was not determined. Here we examine the effects of driving expression of mutant Huntingtin in Olfactory Receptor Neurons (ORNs) by using a marker for cleaved caspase activity to monitor neuronal apoptosis as a function of age. We find widespread caspase activity in various brain regions over time, demonstrating that non-cell autonomous damage is widespread. Improved understanding of which neurons are most vulnerable and why should be useful in developing treatment strategies for neurodegenerative diseases that involve transcellular spreading of aggregates.

  7. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

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    Hoop, Cody L.; Lin, Hsiang-Kai; Kar, Karunakar; Magyarfalvi, Gábor; Lamley, Jonathan M.; Boatz, Jennifer C.; Mandal, Abhishek; Lewandowski, Józef R.; Wetzel, Ronald

    2016-01-01

    Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin–containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand–based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of “intrinsic” polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms. PMID:26831073

  8. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

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    MacDonald Marcy E

    2005-01-01

    Full Text Available Abstract Background Huntington's disease (HD is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that

  9. CCG polymorphisms in the huntingtin gene have no effect on the pathogenesis of patients with Huntington's disease in mainland Chinese families.

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    Zhang, Bao-rong; Tian, Jun; Yan, Ya-ping; Yin, Xin-zhen; Zhao, Guo-hua; Wu, Zhi-ying; Gu, Wei-hong; Xia, Kun; Tang, Bei-sha

    2012-01-15

    Huntington's disease (HD) is caused by the abnormal expansion of CAG repeats in the huntingtin gene (HTT). The adjacent proline-rich region, which also has a CCG polymorphism among people of different races, may also affect the pathogenesis of HD. To study the effect of this polymorphism on patients with HD in mainland China, 53 HD mutant alleles were examined. The results showed that 54.72% of the HD mutant alleles had 10-repeat alleles, and the remaining 45.28% had 7-repeat alleles. Moreover, comparison of the clinical features between the two groups revealed no significant difference. We also investigated its effect on the aggregates in vitro. No significant difference was detected when the morphology and size of the aggregates with the two polymorphisms was compared in cells. Given these findings, it was quite reasonable to suppose that the CCG polymorphism may not influence the pathogenesis of patients with HD in mainland China.

  10. Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis

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    Kordasiewicz, Holly B.; Stanek, Lisa M.; Wancewicz, Edward V.; Mazur, Curt; McAlonis, Melissa M.; Pytel, Kimberly A.; Artates, Jonathan W.; Weiss, Andreas; Cheng, Seng H.; Shihabuddin, Lamya S.; Hung, Gene; Bennett, C. Frank; Cleveland, Don W.

    2012-01-01

    SUMMARY The primary cause of Huntington’s disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebral spinal fluid of symptomatic HD mouse models not only delays disease progression, but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into non-human primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis. PMID:22726834

  11. Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

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    Wüstner Daniel

    2012-11-01

    Full Text Available Abstract Background Fluorescence loss in photobleaching (FLIP is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp function is fitted to fluorescence loss (FL inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP, we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ disease proteins like mutant huntingtin (mtHtt can form large aggregates called inclusion bodies (IB’s. The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and

  12. Natural osmolytes remodel the aggregation pathway of mutant huntingtin exon 1.

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    Borwankar, Tejas; Röthlein, Christoph; Zhang, Gong; Techen, Anne; Dosche, Carsten; Ignatova, Zoya

    2011-03-29

    In response to stress small organic compounds termed osmolytes are ubiquitously accumulated in all cell types to regulate the intracellular solvent quality and to counteract the deleterious effect on the stability and function of cellular proteins. Given the evidence that destabilization of the native state of a protein either by mutation or by environmental changes triggers the aggregation in the neurodegenerative pathologies, the modulation of the intracellular solute composition with osmolytes is an attractive strategy to stabilize an aggregating protein. Here we report the effect of three natural osmolytes on the in vivo and in vitro aggregation landscape of huntingtin exon 1 implicated in the Huntington's disease. Trimethylamine N-oxide (TMAO) and proline redirect amyloid fibrillogenesis of the pathological huntingtin exon 1 to nonamyloidogenic amorphous assemblies via two dissimilar molecular mechanisms. TMAO causes a rapid formation of bulky amorphous aggregates with minimally exposed surface area, whereas proline solubilizes the monomer and suppresses the accumulation of early transient aggregates. Conversely, glycine-betaine enhances fibrillization in a fashion reminiscent of the genesis of functional amyloids. Strikingly, none of the natural osmolytes can completely abrogate the aggregate formation; however, they redirect the amyloidogenesis into alternative, nontoxic aggregate species. Our study reveals new insights into the complex interactions of osmoprotectants with polyQ aggregates.

  13. Coupling of D2R Short but not D2R Long receptor isoform to the Rho/ROCK signaling pathway renders striatal neurons vulnerable to mutant huntingtin.

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    Galan-Rodriguez, Beatriz; Martin, Elodie; Brouillet, Emmanuel; Déglon, Nicole; Betuing, Sandrine; Caboche, Jocelyne

    2017-01-01

    Huntington's disease, an inherited neurodegenerative disorder, results from abnormal polyglutamine extension in the N-terminal region of the huntingtin protein. This mutation causes preferential degeneration of striatal projection neurons. We previously demonstrated, in vitro, that dopaminergic D2 receptor stimulation acted in synergy with expanded huntingtin to increase aggregates formation and striatal death through activation of the Rho/ROCK signaling pathway. In vivo, in a lentiviral-mediated model of expanded huntingtin expression in the rat striatum, we found that the D2 antagonist haloperidol protects striatal neurons against expanded huntingtin-mediated toxicity. Two variant transcripts are generated by alternative splicing of the of D2 receptor gene, the D2R-Long and the D2R-Short, which are thought to play different functional roles. We show herein that overexpression of D2R-Short, but not D2R-Long in cell lines is associated with activation of the RhoA/ROCK signaling pathway. In striatal neurons in culture, the selective D2 agonist Quinpirole triggers phosphorylation of cofilin, a downstream effector of ROCK, which is abrogated by siRNAs that knockdown both D2R-Long and D2R-Short, but not by siRNAs targeting D2R-Long alone. Aggregate formation and neuronal death induced by expanded huntingtin, were potentiated by Quinpirole. This D2 agonist-mediated effect was selectively inhibited by the siRNA targeting both D2R-Long and D2R-Short but not D2R-Long alone. Our data provide evidence for a specific coupling of D2R-Short to the RhoA/ROCK/cofilin pathway, and its involvement in striatal vulnerability to expanded huntingtin. A new route for targeting Rho-ROCK signaling in Huntington's disease is unraveled with our findings.

  14. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

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    Niels H Skotte

    Full Text Available Huntington disease (HD is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs. We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  15. Social defeat: impact on fear extinction and amygdala-prefrontal cortical theta synchrony in 5-HTT deficient mice.

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    Venu Narayanan

    Full Text Available Emotions, such as fear and anxiety, can be modulated by both environmental and genetic factors. One genetic factor is for example the genetically encoded variation of the serotonin transporter (5-HTT expression. In this context, the 5-HTT plays a key role in the regulation of central 5-HT neurotransmission, which is critically involved in the physiological regulation of emotions including fear and anxiety. However, a systematic study which examines the combined influence of environmental and genetic factors on fear-related behavior and the underlying neurophysiological basis is missing. Therefore, in this study we used the 5-HTT-deficient mouse model for studying emotional dysregulation to evaluate consequences of genotype specific disruption of 5-HTT function and repeated social defeat for fear-related behaviors and corresponding neurophysiological activities in the lateral amygdala (LA and infralimbic region of the medial prefrontal cortex (mPFC in male 5-HTT wild-type (+/+, homo- (-/- and heterozygous (+/- mice. Naive males and experienced losers (generated in a resident-intruder paradigm of all three genotypes, unilaterally equipped with recording electrodes in LA and mPFC, underwent a Pavlovian fear conditioning. Fear memory and extinction of conditioned fear was examined while recording neuronal activity simultaneously with fear-related behavior. Compared to naive 5-HTT+/+ and +/- mice, 5-HTT-/- mice showed impaired recall of extinction. In addition, 5-HTT-/- and +/- experienced losers showed delayed extinction learning and impaired recall of extinction. Impaired behavioral responses were accompanied by increased theta synchronization between the LA and mPFC during extinction learning in 5-HTT-/- and +/- losers. Furthermore, impaired extinction recall was accompanied with increased theta synchronization in 5-HTT-/- naive and in 5-HTT-/- and +/- loser mice. In conclusion, extinction learning and memory of conditioned fear can be modulated

  16. Striatal infusion of glial conditioned medium diminishes huntingtin pathology in r6/1 mice.

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    Juan Perucho

    Full Text Available Huntington's disease is a neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin gene which produces widespread neuronal and glial pathology. We here investigated the possible therapeutic role of glia or glial products in Huntington's disease using striatal glial conditioned medium (GCM from fetus mice (E16 continuously infused for 15 and 30 days with osmotic minipumps into the left striatum of R6/1 mice. Animals infused with GCM had significantly less huntingtin inclusions in the ipsilateral cerebral cortex and in the ipsilateral and contralateral striata than mice infused with cerebrospinal fluid. The numbers of DARPP-32 and TH positive neurons were also greater in the ipsilateral but not contralateral striata and substantia nigra, respectively, suggesting a neuroprotective effect of GCM on efferent striatal and nigro-striatal dopamine neurons. GCM increases activity of the autophagic pathway, as shown by the reduction of autophagic substrate, p-62, and the augmentation of LC3 II, Beclin-1 and LAMP-2 protein levels, direct markers of autophagy, in GCM infused mice. GCM also increases BDNF levels. These results suggest that CGM should be further explored as a putative neuroprotective agent in Huntington's disease.

  17. Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington's Disease T Lymphocytes.

    Science.gov (United States)

    Miller, James R C; Träger, Ulrike; Andre, Ralph; Tabrizi, Sarah J

    2015-01-01

    Huntington's disease is a fatal neurodegenerative condition caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system is dysregulated in Huntington's disease and may contribute to its pathogenesis. However, it is not clear whether or to what extent the adaptive immune system is also involved. Here, we carry out the first comprehensive investigation of human ex vivo T lymphocytes in Huntington's disease, focusing on the frequency of a range of T lymphocyte subsets, as well as analysis of proliferation, cytokine production and gene transcription. In contrast to the innate immune system, the intrinsic phenotype of T lymphocytes does not appear to be affected by the presence of mutant huntingtin, with Huntington's disease T lymphocytes exhibiting no significant functional differences compared to control cells. The transcriptional profile of T lymphocytes also does not appear to be significantly affected, suggesting that peripheral immune dysfunction in Huntington's disease is likely to be mediated primarily by the innate rather than the adaptive immune system. This study increases our understanding of the effects of Huntington's disease on peripheral tissues, while further demonstrating the differential effects of the mutant protein on different but related cell types. Finally, this study suggests that the potential use of novel therapeutics aimed at modulating the Huntington's disease innate immune system should not be extended to include the adaptive immune system.

  18. Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington's Disease T Lymphocytes.

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    James R C Miller

    Full Text Available Huntington's disease is a fatal neurodegenerative condition caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system is dysregulated in Huntington's disease and may contribute to its pathogenesis. However, it is not clear whether or to what extent the adaptive immune system is also involved. Here, we carry out the first comprehensive investigation of human ex vivo T lymphocytes in Huntington's disease, focusing on the frequency of a range of T lymphocyte subsets, as well as analysis of proliferation, cytokine production and gene transcription. In contrast to the innate immune system, the intrinsic phenotype of T lymphocytes does not appear to be affected by the presence of mutant huntingtin, with Huntington's disease T lymphocytes exhibiting no significant functional differences compared to control cells. The transcriptional profile of T lymphocytes also does not appear to be significantly affected, suggesting that peripheral immune dysfunction in Huntington's disease is likely to be mediated primarily by the innate rather than the adaptive immune system. This study increases our understanding of the effects of Huntington's disease on peripheral tissues, while further demonstrating the differential effects of the mutant protein on different but related cell types. Finally, this study suggests that the potential use of novel therapeutics aimed at modulating the Huntington's disease innate immune system should not be extended to include the adaptive immune system.

  19. Association of VNTR polymorphisms in DRD4, 5-HTT and DAT1 genes with obesity.

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    Uzun, Mustafa; Saglar, Emel; Kucukyildirim, Sibel; Erdem, Beril; Unlu, Hande; Mergen, Hatice

    2015-05-01

    To investigate the association between VNTR polymorphisms of DRD4, DAT1 and 5-HTT genes and obesity. Peripheral blood samples of 234 obese (BMI ≥ 30) and 148 healthy individuals (BMI ≤ 25) were objected to PCR to detect the VNTR of the 2nd intron of 5-HTT, 3rd exon of DRD4 and 3'UTR of DAT1 genes. The association between obesity and genotype distributions of 5-HTT, DAT1 and DRD4 genes and between obesity and distributions of allele frequencies were tested by Chi Square (χ(2)) test and were not found statistically significant. BMI values for genotype of obese and morbidly obese (BMI > 40) individuals were analyzed by Kruskal-Wallis and not found statistically significant differences between BMI values for the most frequent genotypes of 5-HTT, DAT1 and DRD4 genes. As a conclusion, there was no association between 5-HTT, DAT1 and DRD4 genes VNTR polymorphisms and obesity.

  20. Aggression and 5HTT polymorphism in females: study of synchronized swimming and control groups.

    Science.gov (United States)

    Sysoeva, Olga V; Maluchenko, Natalia V; Timofeeva, Marina A; Portnova, Galina V; Kulikova, Maria A; Tonevitsky, Alexandr G; Ivanitsky, Alexey M

    2009-05-01

    Aggression is a heterogeneous heritable psychological trait, also influenced by environmental factors. Previous studies, mostly conducted on male population, have found some associations of the aggression with the polymorphisms of genes, regulating the activity of serotonin (5-HT) in the brain. However, psychological as well as biochemical manifestations of the aggression are different in males and females. Our study aimed to investigate the association of 5-HTT gene polymorphism with different facets of aggression (BDHI) in females. Two groups: the synchronized swimming and non-athlete control, - were examined to study the possible modulation effect of sport on the association between 5-HTT gene polymorphism and aggression. It was found that in both groups the low-active 5-HTT polymorphism (SS) was associated with increased scores on Indirect Hostility scale and decreased scores on Negativism scale, compared to LL genotype. No interaction effect between sport and 5-HTT polymorphism was found. The higher percentage of LL-carriers and lower of LS-carriers in the synchronized swimming group compared to the control one was observed. This may be the sign of the importance of LL polymorphism of 5-HTT gene, previously associated with higher resistance to stress factors, for being an athlete, although this result has to be taken cautiously keeping in mind the stratification problem. Synchronized swimmers had lower scores on Assault, Negativism, Irritability and Verbal Hostility compared to age-matched control girls (in general and for each 5-HTT genotype separately), suggesting that they may have more matured emotional system (older control group has also lower scores on these scales).

  1. The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of Huntington's disease

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    Frentzel Stefan

    2010-06-01

    Full Text Available Abstract Background Huntington's disease (HD is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD. In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting drug everolimus in the R6/2 mouse model of HD. Results Everolimus decreased phosphorylation of the mTOR target protein S6 kinase indicating brain penetration. However, everolimus did not activate brain macroautophagy as measured by LC3B Western blot analysis. Everolimus protected against early declines in motor performance; however, we found no evidence for neuroprotection as determined by brain pathology. In muscle but not brain, everolimus significantly decreased soluble mutant huntingtin levels. Conclusions Our data suggests that beneficial behavioral effects of everolimus in R6/2 mice result primarily from effects on muscle. Even though everolimus significantly modulated its target brain S6 kinase, this did not decrease mutant huntingtin levels or provide neuroprotection.

  2. Normal and mutant HTT interact to affect clinical severity and progression in Huntington disease

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    Aziz, N A; Jurgens, C K; Landwehrmeyer, G B;

    2009-01-01

    OBJECTIVE: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the HD gene (HTT). We aimed to assess whether interaction between CAG repeat sizes in the mutant and normal allele could affect disease severity and progression. METHODS: Using...... with less severe symptoms and pathology. CONCLUSIONS: Increasing CAG repeat size in normal HTT diminishes the association between mutant CAG repeat size and disease severity and progression in Huntington disease. The underlying mechanism may involve interaction of the polyglutamine domains of normal...

  3. Aberrantly spliced HTT, a new player in Huntington’s disease pathogenesis

    OpenAIRE

    Gipson, Theresa A.; Neueder, Andreas; Wexler, Nancy S.; Bates, Gillian P.; Housman, David

    2013-01-01

    Huntington’s disease (HD) is an adult-onset neurodegenerative disorder caused by a mutated CAG repeat in the huntingtin gene that is translated into an expanded polyglutamine tract. The clinical manifestation of HD is a progressive physical, cognitive, and psychiatric deterioration that is eventually fatal.

  4. Huntington's disease induced cardiac amyloidosis is reversed by modulating protein folding and oxidative stress pathways in the Drosophila heart.

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    Girish C Melkani

    Full Text Available Amyloid-like inclusions have been associated with Huntington's disease (HD, which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102. Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric

  5. Platelet serotonin transporter (5HTt): physiological influences on kinetic characteristics in a large human population.

    Science.gov (United States)

    Banović, Miroslav; Bordukalo-Niksić, Tatjana; Balija, Melita; Cicin-Sain, Lipa; Jernej, Branimir

    2010-01-01

    The present study had two goals: first, to give a detailed description of a reliable method for full kinetic analysis of serotonin transporter (5HTt) on the membrane of human platelets, and second, as a main issue, to report on physiological influences on kinetic characteristics of this transmembrane transport on a large population of healthy individuals. Full kinetic analyses of platelet serotonin uptake were performed on 334 blood donors of both sexes by the use of 14C-radioisotopic method, which was first optimized according to assumptions of enzyme kinetic analyses, with regard to platelet concentration, duration of uptake, concentration of substrate as well as important technical parameters (underpressure of filtration, blanks, incubating temperature, etc). Kinetic parameters of platelet serotonin uptake in the whole population were for V(max): 142 +/- 25.3 pmol 5HT/10(8) platelets/minute and for K(m): 0.404 +/- 0.089 microM 5HT. Besides the report on kinetic values of 5HT transporter protein, we have also described major physiological influences on the mentioned parameters, V(max), K(m) and their derivative, V(max)/K(m) (transporter efficiency): range and frequency distribution of normal values, intraindividual stability over time, lack of age influence, gender dependence and seasonal variations. The report on kinetic values and main physiological influences on platelet serotonin transport kinetics, obtained by the use of thoroughly reassessed methodology, and on by far the largest human population studied until now, offers a reliable frame of reference for pathophysiological studies of this parameter in various clinical fields.

  6. Living in a dangerous world: the shaping of behavioral profile by early environment and 5-HTT genotype

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    Rebecca S Heiming

    2009-09-01

    Full Text Available Anxiety and anxiety disorders are influenced by both, environmental and genetic factors. One genetic factor under scrutiny for anxiety disorders is the genetically encoded variation of the serotonin transporter (5-HTT. The aim of this study was to elucidate the effects of a threatening environment during early phases of life on anxiety-like (ANX and exploratory behavior (EXP in adult mice, varying in serotonin transporter (5-HTT genotype. For this purpose, pregnant and lactating 5-HTT +/- dams were repeatedly exposed to olfactory cues of unfamiliar adult males by introducing small amounts of soiled bedding to their home cage. These stimuli signal the danger of infanticide and simulate a threatening environment. Control females were treated with neutral bedding. The offspring (5-HTT +/+, +/-, -/- were examined for their ANX and EXP. The main results were: (1 a main effect of genotype existed, with 5-HTT -/- showing higher levels of ANX and lower levels of EXP than 5-HTT +/- and wildtypes. (2 When mothers had lived in a threatening environment, their offspring showed increased ANX and reduced EXP compared to controls. (3 These effects were most pronounced in 5-HTT -/- mice. By applying a new ecologically relevant paradigm we conclude: If 5-HTT +/- mothers live in a threatening environment during pregnancy and lactation, their offspring behavioral profile will, in principle, be shaped in an adaptive way preparing the young for an adverse environment. This process is, however, modulated by 5-HTT genotype, bearing the risk that individuals with impaired serotonergic neurotransmission (5-HTT -/- will develop an exaggerated, potentially pathological level of anxiety from gene x environment interactions.

  7. The personality trait openness is related to cerebral 5-HTT levels

    DEFF Research Database (Denmark)

    Kalbitzer, Jan; Frøkjær, Vibe G.; Erritzøe, David

    2009-01-01

    Potentiation of serotonergic transmission increases cognitive flexibility, but can in other circumstances increase sensitivity to stressful environmental cues. The personality trait Openness to Experience reflects and is also associated with an increased risk for mood disorders. We hypothesized...... that the personality trait has an association with a biomarker of serotonergic transmission, the plasma membrane serotonin transporter (5-HTT). In 50 healthy volunteers, we tested for correlations between scores on the NEO-PI-R scale Openness to Experience and its subscales, and cerebral binding of the 5-HTT selective...... and Openness to Values. The latter subscore was negatively correlated with [11C]DASB binding in all brain regions in which [11C]DASB binding was quantified. Genetic analysis showed that homozygote LA carriers had significantly higher [11C]DASB binding in the caudate nucleus, but no significant differences...

  8. 5HTT is associated with the phenotype psychological flexibility: results from a randomized clinical trial.

    Science.gov (United States)

    Gloster, Andrew T; Gerlach, Alexander L; Hamm, Alfons; Höfler, Michael; Alpers, Georg W; Kircher, Tilo; Ströhle, Andreas; Lang, Thomas; Wittchen, Hans-Ulrich; Deckert, Jürgen; Reif, Andreas

    2015-08-01

    Adaption to changing environments is evolutionarily advantageous. Studies that link genetic and phenotypic expression of flexible adjustment to one's context are largely lacking. In this study, we tested the importance of psychological flexibility, or goal-related context sensitivity, in an interaction between psychotherapy outcome for panic disorder with agoraphobia (PD/AG) and a genetic polymorphism. Given the established role of the 5HTT-LPR polymorphism in behavioral flexibility, we tested whether this polymorphism (short group vs. long group) impacted therapy response as a function of various endophenotypes (i.e., psychological flexibility, panic, agoraphobic avoidance, and anxiety sensitivity). Patients with PD/AG were recruited from a large multicenter randomized controlled clinical trial on cognitive-behavioral therapy. Pre- to post-treatment changes by 5HTT polymorphism were analyzed. 5HTT polymorphism status differentiated pre- to post-treatment changes in the endophenotype psychological flexibility (effect size difference d = 0.4, p < 0.05), but none of the specific symptom-related endophenotypes consistently for both the intent-to-treat sample (n = 228) and the treatment completers (n = 194). Based on the consistency of these findings with existing theory on behavioral flexibility, the specificity of the results across phenotypes, and the consistency of results across analyses (i.e., completer and intent to treat), we conclude that 5HTT polymorphism and the endophenotype psychological flexibility are important variables for the treatment of PD/AG. The endophenotype psychological flexibility may help bridge genetic and psychological literatures. Despite the limitation of the post hoc nature of these analyses, further study is clearly warranted.

  9. Oxytocin receptor (OXTR) and serotonin transporter (5-HTT) genes associated with observed parenting.

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    Bakermans-Kranenburg, Marian J; van Ijzendoorn, Marinus H

    2008-06-01

    Both oxytocin and serotonin modulate affiliative responses to partners and offspring. Animal studies suggest a crucial role of oxytocin in mammalian parturition and lactation but also in parenting and social interactions with offspring. The serotonergic system may also be important through its influence on mood and the release of oxytocin. We examined the role of serotonin transporter (5-HTT) and oxytocin receptor (OXTR) genes in explaining differences in sensitive parenting in a community sample of 159 Caucasian, middle-class mothers with their 2-year-old toddlers at risk for externalizing behavior problems, taking into account maternal educational level, maternal depression and the quality of the marital relationship. Independent genetic effects of 5-HTTLPR SCL6A4 and OXTR rs53576 on observed maternal sensitivity were found. Controlling for differences in maternal education, depression and marital discord, parents with the possibly less efficient variants of the serotonergic (5-HTT ss) and oxytonergic (AA/AG) system genes showed lower levels of sensitive responsiveness to their toddlers. Two-way and three-way interactions with marital discord or depression were not significant. This first study on the role of both OXTR and 5-HTT genes in human parenting points to molecular genetic differences that may be implicated in the production of oxytocin explaining differences in sensitive parenting.

  10. Association of serotonin transporter gene (5HTT) polymorphism and juvenile myoclonic epilepsy: a case-control study.

    Science.gov (United States)

    Esmail, Eman H; Labib, Dalia M; Rabie, Walaa A

    2015-09-01

    Serotonin levels might alter susceptibility to seizures. Serotonin transporter (5HTT) gene polymorphisms were found to be associated with some forms of epilepsy. Here, we attempted to examine an association between 5HTT VNTR allele variants in a serotonin transporter gene and epileptogenesis in juvenile myoclonic epilepsy (JME) cases. We conducted a case-control candidate gene study evaluating the frequencies of 5HTT VNTR allele variants using SYBR green real-time PCR with melting curve analysis in JME patients and healthy subjects. Forty patients with JME were selected from the Epilepsy Outpatient Clinic of Kasr Al Ainy Hospital, Cairo University, who had been classified according to the electroclinical classification of the ILAE. The control group consisted of 40 healthy Egyptian subjects. The less efficient transcriptional genotypes for 5-HTT polymorphisms were more frequent in JME patients (OR 9.33, CI 2.85-30.60; p value epileptogenesis in JME.

  11. Social Defeat: Impact on Fear Extinction and Amygdala-Prefrontal Cortical Theta Synchrony in 5-HTT Deficient Mice

    OpenAIRE

    Venu Narayanan; Heiming, Rebecca S.; Friederike Jansen; Jörg Lesting; Norbert Sachser; Hans-Christian Pape; Thomas Seidenbecher

    2011-01-01

    Emotions, such as fear and anxiety, can be modulated by both environmental and genetic factors. One genetic factor is for example the genetically encoded variation of the serotonin transporter (5-HTT) expression. In this context, the 5-HTT plays a key role in the regulation of central 5-HT neurotransmission, which is critically involved in the physiological regulation of emotions including fear and anxiety. However, a systematic study which examines the combined influence of environmental and...

  12. Huntingtin associated protein 1 is involved with the endocytosis of mature brain derived neurotrophic factor%亨廷顿蛋白相关蛋白1与脑源性神经营养因子的胞吞关系的机制研究

    Institute of Scientific and Technical Information of China (English)

    陈锶; 肖波; 毕方方; 冯莉

    2011-01-01

    Objective To investigate the roles of Huntingtin associated protein 1 ( HAP1 ) in the endocytosis of mature brain derived neurotrophic factor (mBDNF) and the underlying mechanism. Methods PC12 cells were differentiated by NGF and co-transfected with plasmid of HAP1 A-CFP and (or) mBDNF-ds-red. The cells were incubated with either recombinant ds-red-labeled mBDNF, or in combination of sheep anti-BDNF antibodies or rabbit anti-p75NTR. The expression of fluorescence and its intercellular location were determined by laser scanning confocal microscope. Cortical neurons from HAP1 +/+ and HAP1-/- mice at postnatal day 1 were cultured and treated with biotin labeled mBDNF to trigger endocytosis. After 60 minutes, the cells were washed and fixed, followed by immunostaining and confocal imaging. Results Co-transfected PC12 cells expressed partly co-localization of HAPl with mBDNF. Cells transfected with HAPl A-CFP which were incubated with recombinant ds-red-labeled mBDNF showed almost complete co-localization of HAPl with mBDNF. The antibodies to BDNF and p75NTR abolished co-internalization of HAPl with mBDNF. In addition, the labeled mBDNF was detected in almost all HAP1+/+, but not in HAP1-/- cortical neurons. Conclusions HAP1 plays an important role in the endocytosis of mBDNF.%目的 探讨亨廷顿蛋白相关蛋白1(HAP1)与脑源性神经营养因子(mBDNF)胞吞的相关性和可能的机制.方法 神经营养因子(NGF)诱导分化PC12细胞,将荧光质粒HAP1A-CFP和(或)mBDNF-ds-red转染进入细胞,培养48 h后在含有BDNF或p75NTR抗体的培养基中继续培养,激光共聚焦显微镜观察荧光的表达情况及其在细胞中的定位;利用小鼠皮层神经元(正常型和HAP1基因敲除型)在生物素标记mBDNF的培养基中孵育60 min,激光共聚焦显微镜观察皮层神经元免疫荧光的效果.结果 共转染HAP1A-CFP和mBDNF-ds-red质粒的细胞,2种荧光蛋白存在部分共定位34%.共转染的细胞在抗BDNF培养

  13. Effect of Simvastatin on 5-HT and 5-HTT in a Rat Model of Pulmonary Artery Hypertension

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    Xue Jiang

    2015-11-01

    Full Text Available Background/Aims: To investigaterole of serotonin (5-HT and serotonin transporter (5-HTT in a rat model of cigarette smoke-induced pulmonary artery hypertension (PAH and the effect of statins on regulating 5HT and 5-HTT. Methods: A rat model of COPD comorbid with PAH was established by cigarette smoke exposure with or without simvastatin administration. The smoking and the simvastatin plus smoking groups were exposed to cigarette smoke daily, and the latter received simvastatin at 5mg/kg, once a day. After 16 weeks of cigarette smoke exposure, body weight and mean pulmonary arterial pressure (mPAP were measured, bronchoalveolar lavage (BAL was performed, and lung tissues and blood samples were collected to determine cardiopulmonary pathology, physiological indices, blood levelof 5-HT and expression of 5-HTT in the lung. Results: In addition to alveolar structural damage (COPD-like injury, chronic cigarette smoke exposure lead to pulmonary artery remodeling and PAH as evidenced by significant elevation of mPAP, RVHI, WT%and WA%. Cigarette smoke exposure resulted in significant reduction in animal body weight, and simvastatin significantly prevented smoke-induced weight loss. The number of inflammatory cells in BALF was dramatically increased in smoke exposed rats, and simvastatin dampened the number of leukocytes, neutrophils, lymphocytes, and macrophages. In addition, circulating 5-HTand expression of 5-HTT in the lung were significantly increased in the smoked rats compared to control rats, and it was significantly reduced by simvastatin. Alteration of BALF inflammatory cells, 5-HT and 5-HTT was significantly correlated with changes of mPAP, RVHI, WT% and WA%. Conclusions: Cigarette smoke exposure could result in not only COPD, but also PAH, which may attribute to the alteration of blood 5-HT and lung tissue 5-HTT. Simvastatin could significantly inhibited 5-HT and 5-HTT expression, and by which mechanism, it may protect animals from development

  14. 5-HTT deficiency affects neuroplasticity and increases stress sensitivity resulting in altered spatial learning performance in the Morris water maze but not in the Barnes maze.

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    Margherita M Karabeg

    Full Text Available The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-, heterozygous (+/- and wildtype (+/+ mice were subjected to the Barnes maze (BM and the Morris water maze (WM, the latter being discussed as more aversive. Additionally, immediate early gene (IEG expression, hippocampal adult neurogenesis (aN, and blood plasma corticosterone were analyzed. While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN. Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of

  15. 5-HTT deficiency affects neuroplasticity and increases stress sensitivity resulting in altered spatial learning performance in the Morris water maze but not in the Barnes maze.

    Science.gov (United States)

    Karabeg, Margherita M; Grauthoff, Sandra; Kollert, Sina Y; Weidner, Magdalena; Heiming, Rebecca S; Jansen, Friederike; Popp, Sandy; Kaiser, Sylvia; Lesch, Klaus-Peter; Sachser, Norbert; Schmitt, Angelika G; Lewejohann, Lars

    2013-01-01

    The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-), heterozygous (+/-) and wildtype (+/+) mice were subjected to the Barnes maze (BM) and the Morris water maze (WM), the latter being discussed as more aversive. Additionally, immediate early gene (IEG) expression, hippocampal adult neurogenesis (aN), and blood plasma corticosterone were analyzed. While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN. Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of raised

  16. Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse Model of Huntington's Disease.

    Science.gov (United States)

    Fox, Jonathan; Lu, Zhen; Barrows, Lorraine

    2015-11-06

    Huntington's disease (HD) is caused by a trinucleotide CAG repeat in the huntingtin gene (HTT) that results in expression of a polyglutamine-expanded mutant huntingtin protein (mHTT). N-terminal fragments of mHTT accumulate in brain neurons and glia as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD.

  17. Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse Model of Huntington’s Disease

    Science.gov (United States)

    Fox, Jonathan; Lu, Zhen; Barrows, Lorraine

    2015-01-01

    Huntington’s disease (HD) is caused by a trinucleotide CAG repeat in the huntingtin gene (HTT) that results in expression of a polyglutamine-expanded mutant huntingtin protein (mHTT). N-terminal fragments of mHTT accumulate in brain neurons and glia as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD. PMID:26664998

  18. Unbiased gene expression analysis implicates the huntingtin polyglutamine tract in extra-mitochondrial energy metabolism.

    Directory of Open Access Journals (Sweden)

    Jong-Min Lee

    2007-08-01

    Full Text Available The Huntington's disease (HD CAG repeat, encoding a polymorphic glutamine tract in huntingtin, is inversely correlated with cellular energy level, with alleles over approximately 37 repeats leading to the loss of striatal neurons. This early HD neuronal specificity can be modeled by respiratory chain inhibitor 3-nitropropionic acid (3-NP and, like 3-NP, mutant huntingtin has been proposed to directly influence the mitochondrion, via interaction or decreased PGC-1alpha expression. We have tested this hypothesis by comparing the gene expression changes due to mutant huntingtin accurately expressed in STHdh(Q111/Q111 cells with the changes produced by 3-NP treatment of wild-type striatal cells. In general, the HD mutation did not mimic 3-NP, although both produced a state of energy collapse that was mildly alleviated by the PGC-1alpha-coregulated nuclear respiratory factor 1 (Nrf-1. Moreover, unlike 3-NP, the HD CAG repeat did not significantly alter mitochondrial pathways in STHdh(Q111/Q111 cells, despite decreased Ppargc1a expression. Instead, the HD mutation enriched for processes linked to huntingtin normal function and Nf-kappaB signaling. Thus, rather than a direct impact on the mitochondrion, the polyglutamine tract may modulate some aspect of huntingtin's activity in extra-mitochondrial energy metabolism. Elucidation of this HD CAG-dependent pathway would spur efforts to achieve energy-based therapeutics in HD.

  19. Hydrothermal Treatment (HTT) of Microalgae: Evaluation of the Process As Conversion Method in an Algae Biorefinery Concept

    NARCIS (Netherlands)

    Garcia Alba, L.; Torri, C.; Samori, C.; Spek, van der J.J.; Fabbri, D.; Kersten, S.R.A.; Brilman, D.W.F.

    2012-01-01

    The hydrothermal treatment (HTT) technology is evaluated for its potential as a process to convert algae and algal debris into a liquid fuel, within a sustainable algae biorefinery concept in which, next to fuels (gaseous and liquid), high value products are coproduced, nutrients and water are recyc

  20. Hydrothermal Treatment (HTT) of Microalgae: Evaluation of the Process As Conversion Method in an Algae Biorefinery Concept

    NARCIS (Netherlands)

    Garcia Alba, Laura; Torri, C.; Samori, C.; van der Spek, J.J.; Fabbri, D.; Kersten, Sascha R.A.; Brilman, Derk Willem Frederik

    2012-01-01

    The hydrothermal treatment (HTT) technology is evaluated for its potential as a process to convert algae and algal debris into a liquid fuel, within a sustainable algae biorefinery concept in which, next to fuels (gaseous and liquid), high value products are coproduced, nutrients and water are

  1. Insulin and IGF-1 regularize energy metabolites in neural cells expressing full-length mutant huntingtin.

    Science.gov (United States)

    Naia, Luana; Ribeiro, Márcio; Rodrigues, Joana; Duarte, Ana I; Lopes, Carla; Rosenstock, Tatiana R; Hayden, Michael R; Rego, A Cristina

    2016-08-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to the expression of mutant huntingtin. Bioenergetic dysfunction has been described to contribute to HD pathogenesis. Thus, treatment paradigms aimed to ameliorate energy deficits appear to be suitable candidates in HD. In previous studies, we observed protective effects of insulin growth factor-1 (IGF-1) in YAC128 and R6/2 mice, two HD mouse models, whereas IGF-1 and/or insulin halted mitochondrial-driven oxidative stress in mutant striatal cells and mitochondrial dysfunction in HD human lymphoblasts. Here, we analyzed the effect of IGF-1 versus insulin on energy metabolic parameters using striatal cells derived from HD knock-in mice and primary cortical cultures from YAC128 mice. STHdh(Q111/Q111) cells exhibited decreased ATP/ADP ratio and increased phosphocreatine levels. Moreover, pyruvate levels were increased in mutant cells, most probably in consequence of a decrease in pyruvate dehydrogenase (PDH) protein expression and increased PDH phosphorylation, reflecting its inactivation. Insulin and IGF-1 treatment significantly decreased phosphocreatine levels, whereas IGF-1 only decreased pyruvate levels in mutant cells. In a different scenario, primary cortical cultures derived from YAC128 mice also displayed energetic abnormalities. We observed a decrease in both ATP/ADP and phosphocreatine levels, which were prevented following exposure to insulin or IGF-1. Furthermore, decreased lactate levels in YAC128 cultures occurred concomitantly with a decline in lactate dehydrogenase activity, which was ameliorated with both insulin and IGF-1. These data demonstrate differential HD-associated metabolic dysfunction in striatal cell lines and primary cortical cultures, both of which being alleviated by insulin and IGF-1.

  2. Epidemiology of Huntington disease: first post-HTT gene analysis of prevalence in Italy.

    Science.gov (United States)

    Squitieri, F; Griguoli, A; Capelli, G; Porcellini, A; D'Alessio, B

    2016-03-01

    Huntington disease (HD) prevalence shows geographic variability and has been recently updated by taking into account the mutation diagnostic test. In Italy, the last epidemiological estimation was reported well before the HTT gene discovery and the availability of the corresponding genetic test. It reported a prevalence of affected subjects ranging between 2.3 and 4.8/100,000 in some restricted areas of Northern Italy. We have performed a service-based epidemiological analysis in a very restricted geographic area named Molise, where our institutions currently operate and represent the only point of reference for rare neuropsychiatric diseases. The estimated prevalence rate found was 10.85/100,000 (95% confidence interval (CI): 7.20-14.50), remarkably higher than that previously described before the gene test analysis was available, and expected to an increase of an additional 17% by 2030, because of Italian population aging. According to our analysis, we estimate that about 6500 subjects are currently affected by HD in Italy, and that this number will further increase in the next decades because of population aging, variable phenotype penetrance and improved life expectancy.

  3. 5-HTT VNTR多态性与偏头痛关系的系统评价%A systematic review with meta-analysis on the relationship of 5-HTT VNTR and migraine

    Institute of Scientific and Technical Information of China (English)

    王贤琦; 李光明; 柳华; 冯胜刚

    2011-01-01

    背景5羟色胺(5-HT)在偏头痛的发病机制中起着重要作用,但是研究5-HT转运体(5-HTT)基因多态性和偏头痛关系的单个遗传关联研究的结果却不一致.目的使用系统评价方法评价5-HTT可变数目串联重复序列(VNTR)多态性和偏头痛的关系.方法 广泛检索中英文数据库以发现合格研究,使用随机或固定效应模型计算合并比值比(OR值),使用Q检验评估研究之间异质性,Egger's(埃格)检验和漏斗图评估发表偏倚.以家族为基础的关联研究则进行描述性分析.结果 总共4个研究纳入meta分析,发现在所有人群中,5-HTT VNTR Stin2.12等位基因或12/12基因型增加了偏头痛的发病风险(Stin2.12等位基因:OR:1.34,95%CI:1.09~1.64,P=0.006; 12/12基因型:OR:1.55,95%CI:1.17~2.05,P=0.002).结论 现有证据表明,5-HTT VNTR多态性(主要是Stin2.12基因型)增加了偏头痛的发病风险,该结论需大样本研究进一步验证.%Objective Serotonin is known to play an important role in the pathogenesis of migraine, but individual genetic association studies that examine the relationship between polymorphisms of serotonin transporter (5-HTT) gene and migraine have yielded inconsistent results. This study aimed to evaluate the association between 5-HTT VNTR polymorphism and migraine using systematic review with meta-analysis. Methods Relevant studies were identified by searching English and Chinese databases extensively. Allele and genotype frequencies for each included study were extracted. The odds ratio (OR) was calculated using a random-effects or fixed-effects model. Q statistic was used to evaluate homogeneity, and Egger's test and Funnel plot were used to assess publication bias. For family-based association studies. A descriptive analysis was carried out) Results A total of 4 studies were identified for meta-analysis. It was found that the 5-HTT VNTR Stin2.12 allele or 12/12 genotype had an increased risk for migraine in the

  4. Correlation of CAG repeat length between the maternal and paternal allele of the Huntingtin gene: evidence for assortative mating

    Directory of Open Access Journals (Sweden)

    Wassink Tom

    2011-10-01

    Full Text Available Abstract Triplet repeats contribute to normal variation in behavioral traits and when expanded, cause brain disorders. While Huntington's Disease is known to be caused by a CAG triplet repeat in the gene Huntingtin, the effect of CAG repeats on brain function below disease threshold has not been studied. The current study shows a significant correlation between the CAG repeat length of the maternal and paternal allele in the Huntingtin gene among healthy subjects, suggesting assortative mating.

  5. Huntington's Disease and Mitochondria.

    Science.gov (United States)

    Jodeiri Farshbaf, Mohammad; Ghaedi, Kamran

    2017-06-21

    Huntington's disease (HD) as an inherited neurodegenerative disorder leads to neuronal loss in striatum. Progressive motor dysfunction, cognitive decline, and psychiatric disturbance are the main clinical symptoms of the HD. This disease is caused by expansion of the CAG repeats in exon 1 of the huntingtin which encodes Huntingtin protein (Htt). Various cellular and molecular events play role in the pathology of HD. Mitochondria as important organelles play crucial roles in the most of neurodegenerative disorders like HD. Critical roles of the mitochondria in neurons are ATP generation, Ca(2+) buffering, ROS generation, and antioxidant activity. Neurons as high-demand energy cells closely related to function, maintenance, and dynamic of mitochondria. In the most neurological disorders, mitochondrial activities and dynamic are disrupted which associate with high ROS level, low ATP generation, and apoptosis. Accumulation of mutant huntingtin (mHtt) during this disease may evoke mitochondrial dysfunction. Here, we review recent findings to support this hypothesis that mHtt could cause mitochondrial defects. In addition, by focusing normal huntingtin functions in neurons, we purpose mitochondria and Huntingtin association in normal condition. Moreover, mHtt affects various cellular signaling which ends up to mitochondrial biogenesis. So, it could be a potential candidate to decline ATP level in HD. We conclude how mitochondrial biogenesis plays a central role in the neuronal survival and activity and how mHtt affects mitochondrial trafficking, maintenance, integrity, function, dynamics, and hemostasis and makes neurons vulnerable to degeneration in HD.

  6. Molecular mechanisms of early-life stress in 5-Htt deficient mice: Gene x environment interactions and epigenetic programming

    OpenAIRE

    2012-01-01

    Early-life stress has been shown to influence the development of the brain and to increase the risk for psychiatric disorders later in life. Furthermore, variation in the human serotonin transporter (5-HTT, SLC6A4) gene is suggested to exert a modulating effect on the association between early-life stress and the risk for depression. At the basis of these gene x environment (G x E) interactions, epigenetic mechanisms, such as DNA-methylation, seem to represent the primary biological processes...

  7. Motivational, proteostatic and transcriptional deficits precede synapse loss, gliosis and neurodegeneration in the B6.HttQ111/+ model of Huntington’s disease

    Science.gov (United States)

    Bragg, Robert M.; Coffey, Sydney R.; Weston, Rory M.; Ament, Seth A.; Cantle, Jeffrey P.; Minnig, Shawn; Funk, Cory C.; Shuttleworth, Dominic D.; Woods, Emily L.; Sullivan, Bonnie R.; Jones, Lindsey; Glickenhaus, Anne; Anderson, John S.; Anderson, Michael D.; Dunnett, Stephen B.; Wheeler, Vanessa C.; MacDonald, Marcy E.; Brooks, Simon P.; Price, Nathan D.; Carroll, Jeffrey B.

    2017-01-01

    We investigated the appearance and progression of disease-relevant signs in the B6.HttQ111/+ mouse, a genetically precise model of the mutation that causes Huntington’s disease (HD). We find that B6.HttQ111/+ mice are healthy, show no overt signs of central or peripheral inflammation, and no gross motor impairment as late as 12 months of age. Behaviorally, we find that 4–9 month old B6.HttQ111/+ mice have normal activity levels and show no clear signs of anxiety or depression, but do show clear signs of reduced motivation. The neuronal density, neuronal size, synaptic density and number of glia is normal in B6.HttQ111/+ striatum, the most vulnerable brain region in HD, up to 12 months of age. Despite this preservation of the synaptic and cellular composition of the striatum, we observe clear progressive, striatal-specific transcriptional dysregulation and accumulation of neuronal intranuclear inclusions (NIIs). Simulation studies suggest these molecular endpoints are sufficiently robust for future preclinical studies, and that B6.HttQ111/+ mice are a useful tool for modeling disease-modifying or neuroprotective strategies for disease processes before the onset of overt phenotypes. PMID:28176805

  8. 5-HT(1A) receptor and 5-HTT binding during the menstrual cycle in healthy women examined with [(11)C] WAY100635 and [(11)C] MADAM PET.

    Science.gov (United States)

    Jovanovic, Hristina; Karlsson, Per; Cerin, Asta; Halldin, Christer; Nordström, Anna-Lena

    2009-04-30

    The aim of the present study was to explore the effects of the menstrual cycle phases on 5-HT(1A) receptor and 5-HTT binding potentials (BPs) in healthy women by using positron emission tomography (PET). Women were investigated in the follicular and luteal phase of the menstrual cycle with radioligands [(11)C]WAY10035 (n=13) and [(11)C]MADAM (n=8) to study 5-HT(1A) and 5-HTT BPs. The BPs values were quantified using the simplified reference tissue model. The phases of the menstrual cycle were characterized by transvaginal ultrasound (TSV) and plasma levels of hormones estradiol (E(2)), progesterone (P(4)), follicle stimulating hormone (FSH) and luteinizing hormone (LH).The 5-HT(1A) receptor and 5-HTT BPs did not significantly differ between follicular and luteal phases in any of the investigated regions. There were no significant correlations between the change in E(2) or P(4) values with the change in 5-HT(1A) receptor or 5-HTT BPs. The results provide principally a new in vivo finding in human female biology, suggesting the absence of influence of menstrual cycle phase on 5-HT(1A) receptors or 5-HTT. The finding however does not preclude that gonadal hormones differentially influence central serotonin system inwomen and men, which might contribute to gender differences in serotonin-associated disorders.

  9. Huntington's disease

    OpenAIRE

    Bates, G P; Dorsey, R.; Gusella, J F; Hayden, M. R.; Kay, C; Leavitt, B. R.; Nance, M; Ross, C A; Scahill, R. I.; Wetzel, R.; Wild, E. J.; Tabrizi, S.J.

    2015-01-01

    Huntington’s disease is devastating to patients and their families — with autosomal dominant inheritance, onset typically in the prime of adult life, progressive course and combination of motor, cognitive and behavioural features. The disease is caused by an expanded CAG trinucleotide repeat (of variable length) in HTT, the gene which encodes the protein huntingtin. In mutation carriers, huntingtin is produced with abnormally long polyglutamine sequences that confers toxic gains of function a...

  10. Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

    Science.gov (United States)

    Grondin, Richard; Ge, Pei; Chen, Qingmin; Sutherland, Jessica E; Zhang, Zhiming; Gash, Don M; Stiles, David K; Stewart, Gregory R; Sah, Dinah W Y; Kaemmerer, William F

    2015-06-30

    One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

  11. Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA

    Directory of Open Access Journals (Sweden)

    Richard Grondin

    2015-01-01

    Full Text Available One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27–39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

  12. Co-localization of brain-derived neurotrophic factor (BDNF) and wild-type huntingtin in normal and quinolinic acid-lesioned rat brain.

    Science.gov (United States)

    Fusco, Francesca R; Zuccato, Chiara; Tartari, Marzia; Martorana, Alessandro; De March, Zena; Giampà, Carmela; Cattaneo, Elena; Bernardi, Giorgio

    2003-09-01

    Loss of huntingtin-mediated brain-derived neurotrophic factor (BDNF) gene transcription has been described in Huntington's disease (HD) [Zuccato et al. (2001) Science, 293, 493-498]. It has been shown that BDNF is synthesized in the pyramidal layer of cerebral cortex and released in the striatum [Altar et al. (1997) Nature, 389, 856-860; Conner et al. (1997) J. Neurosci., 17, 2295-2313]. Here we show the cellular localization of BDNF in huntingtin-containing neurons in normal rat brain; our double-label immunofluorescence study shows that huntingtin and BDNF are co-contained in approximately 99% of pyramidal neurons of motor cortex. In the striatum, huntingtin is expressed in 75% of neurons containing BDNF. In normal striatum we also show that BDNF is contained in cholinergic and in NOS-containing interneurons, which are relatively resistant to HD degeneration. Furthermore, we show a reduction in huntingtin and in BDNF immunoreactivity in cortical neurons after striatal excitotoxic lesion. Our data are confirmed by an ELISA study of BDNF and by a Western blot analysis of huntingtin in cortex of quinolic acid (QUIN)-lesioned hemispheres. In the lesioned striatum we describe that the striatal subpopulation of cholinergic neurons, surviving degeneration, contain BDNF. The finding that BDNF is contained in nearly all neurons that contain huntingtin in the normal cortex, along with the reduced expression of BDNF after QUIN injection of the striatum, shows that huntingtin may be required for BDNF production in cortex.

  13. Silencing Huntington's chorea: Is RNA Interference a Potential Cure?

    OpenAIRE

    Metz, Gerlinde A.; Whishaw, Ian Q.; Afra Foroud; Nafisa M Jadavji

    2006-01-01

    In 1872, George Huntington described Huntington's disease as characterized by motor, cognitive and psychiatric impairments. Huntington's disease is a dominant and autosomal mutation on chromosome 4 featuring the insertion of numerous CAG repeats. CAG codes for the amino acid, glutmanine that forms part of the Huntingtin protein (htt). Excess glutamine attachments make htt prone to accumulate in neurons. Three genes can be considered when developing therapies for Huntington's disease. They inc...

  14. Impact of Institutional Care on Attachment Disorganization and Insecurity of Ukrainian Preschoolers: Protective Effect of the Long Variant of the Serotonin Transporter Gene (5HTT)

    Science.gov (United States)

    Bakermans-Kranenburg, Marian J.; Dobrova-Krol, Natasha; van IJzendoorn, Marinus

    2012-01-01

    Institutional care has been shown to lead to insecure and disorganized attachments and indiscriminate friendliness. Some children, however, are surprisingly resilient to the adverse environment. Here the protective role of the long variant of the serotonin receptor gene (5HTT) is explored in a small hypothesis-generating study of 37 Ukrainian…

  15. 5-HTT and 5-HT(1A) receptor occupancy of the novel substance vortioxetine (Lu AA21004). A PET study in control subjects.

    Science.gov (United States)

    Stenkrona, Per; Halldin, Christer; Lundberg, Johan

    2013-10-01

    Vortioxetine (Lu AA21004) is a new potential substance for the treatment of anxiety and mood disorders. It has high affinity for the 5-HT transporter (5-HTT) and moderate affinity for the 5-HT1A receptor in vitro. Positron emission tomography (PET) has commonly been used to examine the relation between dose/plasma concentration and occupancy to predict relevant dose intervals in a clinical setting. In this study 11 control subjects were examined with PET and [¹¹C]MADAM at baseline, after a single dose and after 9 days of dosing with Lu AA21004 (2.5, 10 or 60 mg) for quantification of 5-HTT occupancy. Four subjects were examined with PET and [¹¹C]WAY 100635 at baseline, after a single dose and after 9 days of dosing of Lu AA21004 (30 mg) for quantification of 5-HT(1A) occupancy. To allow for quantification of binding in the raphe nuclei, PET data were analyzed using wavelet aided parametric imaging. 5-HTT occupancy ranged from 2 (mean, 2.5 mg day 1) to 97% (60 mg day 9). The apparent affinity of Lu AA21004 binding to 5-HTT (KD(ND)) was calculated to 16.7 nM (R=0.95), and the corresponding oral dose (KD(ND)-dose) to 8.5 mg (R=0.91). No significant occupancy of 5-HT(1A) receptors was found after dosing of 30 mg Lu AA21004. Based on the literature and the present [¹¹C]MADAM binding data, a dose of 20-30 mg Lu AA21004 is suggested to give clinically relevant occupancy of the 5-HTT.

  16. Stoichiometry of base excision repair proteins correlates with increased somatic CAG instability in striatum over cerebellum in Huntington's disease transgenic mice.

    Science.gov (United States)

    Goula, Agathi-Vassiliki; Berquist, Brian R; Wilson, David M; Wheeler, Vanessa C; Trottier, Yvon; Merienne, Karine

    2009-12-01

    Huntington's disease (HD) is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT) gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER) enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5'-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1), the main enzyme responsible for 5'-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP-BER), were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLbeta was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLbeta strand displacement activity during LP-BER promotes the formation of stable 5'-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes is one critical

  17. Stoichiometry of base excision repair proteins correlates with increased somatic CAG instability in striatum over cerebellum in Huntington's disease transgenic mice.

    Directory of Open Access Journals (Sweden)

    Agathi-Vassiliki Goula

    2009-12-01

    Full Text Available Huntington's disease (HD is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5'-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1, the main enzyme responsible for 5'-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP-BER, were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLbeta was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLbeta strand displacement activity during LP-BER promotes the formation of stable 5'-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes

  18. Dysregulation of system xc(-) expression induced by mutant huntingtin in a striatal neuronal cell line and in R6/2 mice.

    Science.gov (United States)

    Frederick, Natalie M; Bertho, Julie; Patel, Kishan K; Petr, Geraldine T; Bakradze, Ekaterina; Smith, Sylvia B; Rosenberg, Paul A

    2014-10-01

    Oxidative stress has been implicated in the pathogenesis of Huntington's disease (HD), however, the origin of the oxidative stress is unknown. System xc(-) plays a role in the import of cystine to synthesize the antioxidant glutathione. We found in the STHdh(Q7/Q7) and STHdh(Q111/Q111) striatal cell lines, derived from neuronal precursor cells isolated from knock-in mice containing 7 or 111 CAG repeats in the huntingtin gene, that there is a decrease in system xc(-) function. System xc(-) is composed of two proteins, the substrate specific transporter, xCT, and an anchoring protein, CD98. The decrease in function in system xc(-) that we observed is caused by a decrease in xCT mRNA and protein expression in the STHdh(Q111/Q111) cells. In addition, we found a decrease in protein and mRNA expression in the transgenic R6/2 HD mouse model at 6weeks of age. STHdh(Q111/Q111) cells have lower basal levels of GSH and higher basal levels of ROS. Acute inhibition of system xc(-) causes greater increase in oxidative stress in the STHdh(Q111/Q111) cells than in the STHdh(Q7/Q7) cells. These results suggest that a defect in the regulation of xCT may be involved in the pathogenesis of HD by compromising xCT expression and increasing susceptibility to oxidative stress.

  19. Lithium Down-regulates Histone Deacetylase 1 (HDAC1) and Induces Degradation of Mutant Huntingtin*

    Science.gov (United States)

    Wu, Shuai; Zheng, Shui-Di; Huang, Hong-Ling; Yan, Li-Chong; Yin, Xiao-Fei; Xu, Hai-Neng; Zhang, Kang-Jian; Gui, Jing-Hua; Chu, Liang; Liu, Xin-Yuan

    2013-01-01

    Lithium is an effective mood stabilizer that has been clinically used to treat bipolar disorder for several decades. Recent studies have suggested that lithium possesses robust neuroprotective and anti-tumor properties. Thus far, a large number of lithium targets have been discovered. Here, we report for the first time that HDAC1 is a target of lithium. Lithium significantly down-regulated HDAC1 at the translational level by targeting HDAC1 mRNA. We also showed that depletion of HDAC1 is essential for the neuroprotective effects of lithium and for the lithium-mediated degradation of mutant huntingtin through the autophagic pathway. Our studies explain the multiple functions of lithium and reveal a novel mechanism for the function of lithium in neurodegeneration. PMID:24165128

  20. Differential susceptibility in spillover between interparental conflict and maternal parenting practices: evidence for OXTR and 5-HTT genes.

    Science.gov (United States)

    Sturge-Apple, Melissa L; Cicchetti, Dante; Davies, Patrick T; Suor, Jennifer H

    2012-06-01

    Guided by the affective spillover hypothesis and the differential susceptibility to environmental influence frameworks, the present study examined how associations between interparental conflict and mothers' parenting practices were moderated by serotonin transporter (5-HTT) and oxytocin receptor (OXTR) genes. A sample of 201 mothers and their 2-year old child participated in a laboratory-based research assessment. Results supported differential susceptibility hypotheses within spillover frameworks. With respect to OXTR rs53576, mothers with the GG genotype showed greater differential maternal sensitivity across varying levels of interparental conflict. Mothers with one or two copies of the 5-HTTLPR S allele demonstrated differential susceptibility for both sensitive and harsh/punitive caregiving behaviors. Finally, analyses examined whether maternal depressive symptoms and emotional closeness to their child mediated the moderating effects. Findings suggest that maternal emotional closeness with their child indirectly linked OXTR with maternal sensitivity. The results highlight how molecular genetics may explain heterogeneity in spillover models with differential implications for specific parenting behaviors. Implications for clinicians and therapists working with maritally distressed parents are discussed.

  1. Association between Neurocognitive Impairment and the Short Allele of the 5-HTT Promoter Polymorphism in Depression: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Hely Kalska

    2013-01-01

    Full Text Available Depression has been shown to be associated with cognitive deficits in various cognitive domains. However, it is still unclear which factors contribute to cognitive impairment. The objective of this study was to find out whether a functional polymorphism in the promoter region of the serotonin transporter (5-HTTLPR gene is associated with the impairment of cognitive functioning among depressed patients. In a pilot study, a sample of 19 patients with major depressive disorder (MDD and 19 healthy controls was investigated with an extensive psychiatric and neuropsychological examination. All participants were genotyped for 5-HTTLPR. Depressed patients with the short allele of the 5-HTT promoter region exhibited inferior cognitive performance compared to patients with the long allele polymorphism. In healthy controls, no association between genotype and cognitive performance was found. The result suggests that in MDD patients with the short allele of the 5-HTTLPR polymorphism the vulnerability to cognitive impairment is increased compared to MDD patients without the short allele inheritance. These preliminary findings need to be confirmed in a larger cohort of MDD patients.

  2. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.;

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

  3. How the cerebral serotonin homeostasis predicts environmental changes: a model to explain seasonal changes of brain 5-HTT as intermediate phenotype of the 5-HTTLPR.

    Science.gov (United States)

    Kalbitzer, Jan; Kalbitzer, Urs; Knudsen, Gitte Moos; Cumming, Paul; Heinz, Andreas

    2013-12-01

    Molecular imaging studies with positron emission tomography have revealed that the availability of serotonin transporter (5-HTT) in the human brain fluctuates over the course of the year. This effect is most pronounced in carriers of the short allele of the 5-HTT promoter region (5-HTTLPR), which has in several previous studies been linked to an increased risk to develop mood disorders. We argue that long-lasting fluctuations in the cerebral serotonin transmission, which is regulated via the 5-HTT, are responsible for mediating responses to environmental changes based on an assessment of the expected "safety" of the environment; this response is obtained in part through serotonergic modulation of the hypothalamic-pituitary-adrenal (HPA) axis. We posit that the intermediate phenotype of the s-allele may properly be understood as mediating a trade-off, wherein increased responsiveness of cerebral serotonin transmission to seasonal and other forms of environmental change imparts greater behavioral flexibility, at the expense of increased vulnerability to stress. This model may explain the somewhat higher prevalence of the s-allele in some human populations dwelling at geographic latitudes with pronounced seasonal climatic changes, while this hypothesis does not rule out that genetic drift plays an additional or even exclusive role. We argue that s-allele manifests as an intermediate phenotype in terms of an increased responsiveness of the 5-HTT expression to number of daylight hours, which may serve as a stable surrogate marker of other environmental factors, such as availability of food and safety of the environment in populations that live closer to the geographic poles.

  4. Multiple discrete soluble aggregates influence polyglutamine toxicity in a Huntington’s disease model system

    Science.gov (United States)

    Xi, Wen; Wang, Xin; Laue, Thomas M.; Denis, Clyde L.

    2016-01-01

    Huntington’s disease (HD) results from expansions of polyglutamine stretches (polyQ) in the huntingtin protein (Htt) that promote protein aggregation, neurodegeneration, and death. Since the diversity and sizes of the soluble Htt-polyQ aggregates that have been linked to cytotoxicity are unknown, we investigated soluble Htt-polyQ aggregates using analytical ultracentrifugation. Soon after induction in a yeast HD model system, non-toxic Htt-25Q and cytotoxic Htt-103Q both formed soluble aggregates 29S to 200S in size. Because current models indicate that Htt-25Q does not form soluble aggregates, reevaluation of previous studies may be necessary. Only Htt-103Q aggregation behavior changed, however, with time. At 6 hr mid-sized aggregates (33S to 84S) and large aggregates (greater than 100S) became present while at 24 hr primarily only mid-sized aggregates (20S to 80S) existed. Multiple factors that decreased cytotoxicity of Htt-103Q (changing the length of or sequences adjacent to the polyQ, altering ploidy or chaperone dosage, or deleting anti-aging factors) altered the Htt-103Q aggregation pattern in which the suite of mid-sized aggregates at 6 hr were most correlative with cytotoxicity. Hence, the amelioration of HD and other neurodegenerative diseases may require increased attention to and discrimination of the dynamic alterations in soluble aggregation processes. PMID:27721444

  5. Evaluation of the Relationship between 5-HTT and MAO Gene Polymorphisms, Mood and Level of Anxiety among Postmenopausal Women

    Directory of Open Access Journals (Sweden)

    Elżbieta Grochans

    2014-12-01

    Full Text Available Objective: The aim of this study was to analyze how mood and anxiety level are related to the functional genetic polymorphism in the promoter region of SLC6A4 (5-HTTLPR and the 30-bp VNTR polymorphism in the MAO A promoter region. Methods: The study involved 272 postmenopausal women from Poland. The authors employed the State-Trait Anxiety Inventory for measuring levels of anxiety, the Mood Adjective Check List for measuring mood, and genetic tests. Results: Analysis did not show any statistically significant differences in the mean levels of anxiety, and mood disorders in women in relation to genotypes of the 5-HTTLPR (SLC6A4 polymorphism and the 30-bp VNTR polymorphism in the MAO A promoter region. However, these problems were more severe among women with s/s genotype. In the case of MAO A gene polymorphism, the level of anxiety was higher in women with a 4/4 genotype. Conclusions: The study did not prove the possibility of the identification of homogeneous groups of women with an elevated risk of developing anxiety and mood disorders during the post-menopausal period. Nevertheless, it showed that respondents with s/s genotype of the 44-bp polymorphism in the 5-HTT (SLC6A4 promoter region had the highest average anxiety levels both as a state and as a trait. Furthermore, the analysis of the 30-bp VNTR polymorphism in the MAO A promoter region demonstrated slight differences in anxiety levels between the women, indicating that those with a 4/4 genotype had higher severity of anxiety symptoms.

  6. REVIEW-ARTICLE Intermediate alleles of Huntington's disease HTT gene in different populations worldwide: a systematic review.

    Science.gov (United States)

    Apolinário, T A; Paiva, C L A; Agostinho, L A

    2017-04-05

    Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by a dynamic mutation due to the expansion of CAG repeats in the HTT gene (4p16.3). The considered normal alleles have less than 27 CAG repeats. Intermediate alleles (IAs) show 27 to 35 CAG repeats and expanded alleles have more than 35 repeats. The IAs apparently have shown a normal phenotype. However, there are some reported associations between individuals that bear an IA and clinical HD signs, such as behavioral disturbs. The association of IAs with the presence of clinical signs gives clinical relevance to these patients. We emphasized the importance of determining the frequency of IA alleles in the general population as well as in HD families. Therefore, the aim of this study was to conduct a systematic review, in order to investigate the frequency of IAs in the overall chromosomes of different ethnic groups and of families with HD history worldwide as well as the frequency of individuals who bear the intermediate alleles. We searched indexed articles from the following electronic databases: U.S. National Library of Medicine and the National Institutes of Health (PubMed), Pubmed Central (PMC) and Virtual Health Library (VHL). Therefore, 488 articles were obtained and, of these, 33 had been published in more than one database. We accepted the article of only one database and ended up with 455 articles for this review. The frequency of IAs within the chromosomes of the general population ranged from 0.45 to 8.7% and of individuals with family history of HD ranged from 0.05 to 5.1%. The higher frequency of IAs in the general population (8.7%) was found in one Brazilian cohort.

  7. Evaluation of the Relationship between 5-HTT and MAO Gene Polymorphisms, Mood and Level of Anxiety among Postmenopausal Women

    Science.gov (United States)

    Grochans, Elżbieta; Jurczak, Anna; Szkup, Małgorzata; Samochowiec, Agnieszka; Włoszczak-Szubzda, Anna; Karakiewicz, Beata; Grzywacz, Anna; Brodowska, Agnieszka; Samochowiec, Jerzy

    2014-01-01

    Objective: The aim of this study was to analyze how mood and anxiety level are related to the functional genetic polymorphism in the promoter region of SLC6A4 (5-HTTLPR) and the 30-bp VNTR polymorphism in the MAO A promoter region. Methods: The study involved 272 postmenopausal women from Poland. The authors employed the State-Trait Anxiety Inventory for measuring levels of anxiety, the Mood Adjective Check List for measuring mood, and genetic tests. Results: Analysis did not show any statistically significant differences in the mean levels of anxiety, and mood disorders in women in relation to genotypes of the 5-HTTLPR (SLC6A4) polymorphism and the 30-bp VNTR polymorphism in the MAO A promoter region. However, these problems were more severe among women with s/s genotype. In the case of MAO A gene polymorphism, the level of anxiety was higher in women with a 4/4 genotype. Conclusions: The study did not prove the possibility of the identification of homogeneous groups of women with an elevated risk of developing anxiety and mood disorders during the post-menopausal period. Nevertheless, it showed that respondents with s/s genotype of the 44-bp polymorphism in the 5-HTT (SLC6A4) promoter region had the highest average anxiety levels both as a state and as a trait. Furthermore, the analysis of the 30-bp VNTR polymorphism in the MAO A promoter region demonstrated slight differences in anxiety levels between the women, indicating that those with a 4/4 genotype had higher severity of anxiety symptoms. PMID:25547397

  8. Regulation of P-glycoprotein expression in brain capillaries in Huntington's disease and its impact on brain availability of antipsychotic agents risperidone and paliperidone.

    Science.gov (United States)

    Kao, Yu-Han; Chern, Yijuang; Yang, Hui-Ting; Chen, Hui-Mei; Lin, Chun-Jung

    2016-08-01

    Huntington's disease (HD) is a neurodegenerative disease marked by an expanded polyglutamine (polyQ) tract on the huntingtin (HTT) protein that may cause transcriptional dysfunction. This study aimed to investigate the regulation and function of P-glycoprotein, an important efflux transporter, in brain capillaries in HD. The results showed that, compared with the littermate controls, R6/2 HD transgenic mice with the human mutant HTT gene had higher levels of P-glycoprotein mRNA and protein and enhanced NF-κB activity in their brain capillaries. Higher P-glycoprotein expression was also observed in the brain capillaries of human HD patients. Consistent with this enhanced P-glycoprotein expression, brain extracellular levels and brain-to-plasma ratios of the antipsychotic agents risperidone and paliperidone were significantly lower in R6/2 mice than in their littermate controls. Exogenous expression of human mutant HTT protein with expanded polyQ (mHTT-109Q) in HEK293T cells enhanced the levels of P-glycoprotein transcripts and NF-κB activity compared with cells expressing normal HTT-25Q. Treatment with the IKK inhibitor, BMS-345541, decreased P-glycoprotein mRNA level in cells transfected with mHTT-109Q or normal HTT-25Q In conclusion, mutant HTT altered the expression of P-glycoprotein through the NF-κB pathway in brain capillaries in HD and markedly affected the availability of P-glycoprotein substrates in the brain.

  9. Reciprocal Efficiency of RNQ1 and Polyglutamine Detoxification in the Cytosol and Nucleus

    OpenAIRE

    Douglas, Peter M.; Summers, Daniel W.; Ren, Hong-Yu; Cyr, Douglas M.

    2009-01-01

    Onset of proteotoxicity is linked to change in the subcellular location of proteins that cause misfolding diseases. Yet, factors that drive changes in disease protein localization and the impact of residence in new surroundings on proteotoxicity are not entirely clear. To address these issues, we examined aspects of proteotoxicity caused by Rnq1-green fluorescent protein (GFP) and a huntingtin's protein exon-1 fragment with an expanded polyglutamine tract (Htt-103Q), which is dependent upon t...

  10. Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington's disease knock-in mice.

    Science.gov (United States)

    Lloret, Alejandro; Dragileva, Ella; Teed, Allison; Espinola, Janice; Fossale, Elisa; Gillis, Tammy; Lopez, Edith; Myers, Richard H; MacDonald, Marcy E; Wheeler, Vanessa C

    2006-06-15

    Genetically precise models of Huntington's disease (HD), Hdh CAG knock-in mice, are powerful systems in which phenotypes associated with expanded HD CAG repeats are studied. To dissect the genetic pathways that underlie such phenotypes, we have generated Hdh(Q111) knock-in mouse lines that are congenic for C57BL/6, FVB/N and 129Sv inbred genetic backgrounds and investigated four Hdh(Q111) phenotypes in these three genetic backgrounds: the intergenerational instability of the HD CAG repeat and the striatal-specific somatic HD CAG repeat expansion, nuclear mutant huntingtin accumulation and intranuclear inclusion formation. Our results reveal increased intergenerational and somatic instability of the HD CAG repeat in C57BL/6 and FVB/N backgrounds compared with the 129Sv background. The accumulation of nuclear mutant huntingtin and the formation of intranuclear inclusions were fastest in the C57BL/6 background, slowest in the 129Sv background and intermediate in the FVB/N background. Inbred strain-specific differences were independent of constitutive HD CAG repeat size and did not correlate with Hdh mRNA levels. These data provide evidence for genetic modifiers of both intergenerational HD CAG repeat instability and striatal-specific phenotypes. Different relative contributions of C57BL/6 and 129Sv genetic backgrounds to the onset of nuclear mutant huntingtin and somatic HD CAG repeat expansion predict that the initiation of each of these two phenotypes is modified by different genes. Our findings set the stage for defining disease-related genetic pathways that will ultimately provide insight into disease mechanism.

  11. Mutant huntingtin activates Nrf2-responsive genes and impairs dopamine synthesis in a PC12 model of Huntington's disease

    Directory of Open Access Journals (Sweden)

    den Dunnen Johan T

    2008-10-01

    Full Text Available Abstract Background Huntington's disease is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion in the HD or Huntington's disease gene. Although micro array studies on patient and animal tissue provide valuable information, the primary effect of mutant huntingtin will inevitably be masked by secondary processes in advanced stages of the disease. Thus, cell models are instrumental to study early, direct effects of mutant huntingtin. mRNA changes were studied in an inducible PC12 model of Huntington's disease, before and after aggregates became visible, to identify groups of genes that could play a role in the early pathology of Huntington's disease. Results Before aggregation, up-regulation of gene expression predominated, while after aggregates became visible, down-regulation and up-regulation occurred to the same extent. After aggregates became visible there was a down-regulation of dopamine biosynthesis genes accompanied by down-regulation of dopamine levels in culture, indicating the utility of this model to identify functionally relevant pathways. Furthermore, genes of the anti-oxidant Nrf2-ARE pathway were up-regulated, possibly as a protective mechanism. In parallel, we discovered alterations in genes which may result in increased oxidative stress and damage. Conclusion Up-regulation of gene expression may be more important in HD pathology than previously appreciated. In addition, given the pathogenic impact of oxidative stress and neuroinflammation, the Nrf2-ARE signaling pathway constitutes a new attractive therapeutic target for HD.

  12. Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and β-cell apoptosis

    DEFF Research Database (Denmark)

    Berchtold, Lukas Adrian; Størling, Zenia Marian; Ortis, Fernanda

    2011-01-01

    Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing...

  13. [The influence of parents personality and DRD4 and 5HTT genes polymorphisms on predisposition to alcohol dependence in their sons].

    Science.gov (United States)

    Samochowiec, Agnieszka; Horodnicki, Jan M; Samochowiec, Jerzy

    2011-01-01

    The aim of this work was to investigate differences in parents' personality and dopaminergic and serotoninergic gene polymorphisms which may affect certain predispositions to alcohol dependence as described in the typology developed by R. Cloninger and O.M. Lesch. Also the possibility of recognising their genotypes DRD4 (Gene ID: 1815A ) and 5HTT (Gene ID: 6532) could be helpful in predicting predisposition to addiction. A total number of 213 individuals (71 Polish trios), Caucasian families were investigated. Fathers' mean age was: 61.7 +/- 10.8 and mothers were 59 +/- 10 years old. None of the parents fulfilled the criteria of alcohol dependence. The alcohol dependent probands were male, with confirmed biological descent, mean age: 35.2 +/- 9.7 years. In all the participants TCI was performed. Characterisation of alcohol dependence and the course of withdrawal were obtained by SSAGA. Specially designed questionnaires based on Cloninger and Lesch typologies were used. The essential data on both parents was collected and AUDIT was performed. DRD4 and 5HTT gene polymorphisms were determined by PCR and TDT test was calculated. TDT analysis showed no differences in the transmission of alleles of 5HTT and DRD4 genes in the investigated families. The analysis of TCI personality profiles confirmed no statistically significant relations between Cloninger 1 and 2 subtypes of alcoholics. A statistically significant difference was recorded between the scores for groups I and II classified according to Lesch's typology in dimensions NS, NS2 and NS4. Fathers of probands characterised as type I according to Cloninger had statistically lower scores in dimension C and C5 in comparison to type II fathers. Fathers of type II alcoholics according to Lesch's typology had higher NS2. Mothers of type I alcoholics according to Cloninger had statistically lower scores in dimension HA2 in comparison with type II mothers. On the basis of the above presented findings it can be stated that

  14. Differential vulnerability of neurons in Huntington's disease: The role of cell type-specific features

    Science.gov (United States)

    Han, Ina; You, YiMei; Kordower, Jeffrey H.; Brady, Scott T.; Morfini, Gerardo A.

    2010-01-01

    Abnormal expansion of a polyglutamine tract in huntingtin (Htt) protein results in Huntington's disease (HD), an autosomal dominant neurodegenerative disorder involving progressive loss of motor and cognitive function. Contrasting with the ubiquitous tissue expression of polyglutamine-expanded Htt (polyQ-Htt), HD pathology is characterized by the increased vulnerability of specific neuronal populations within the striatum and the cerebral cortex. Morphological, biochemical, and functional characteristics of neurons affected in HD that might render these cells more vulnerable to the toxic effects of polyQ-Htt are covered in this review. The differential vulnerability of neurons observed in HD is discussed in the context of various major pathogenic mechanisms proposed to date, and in line with evidence showing a “dying-back” pattern of degeneration in affected neuronal populations. PMID:20236390

  15. Hunting for huntingtin associated factors: Identification and characterization of huntingtin expanded polyglutamine aggregate associated factors and their impact on Huntington disease model cellular toxicity

    Science.gov (United States)

    2016-05-20

    medical economics 16:1043-50 47. Doi H, Mitsui K, Kurosawa M, Machida Y, Kuroiwa Y, Nukina N. 2004. Identification of ubiquitin-interacting proteins in...biological chemistry 285:26304-14 60. Furukawa Y, Kaneko K, Matsumoto G, Kurosawa M, Nukina N. 2009. Cross- seeding fibrillation of Q/N-rich proteins offers

  16. Life satisfaction in the new country: a multilevel longitudinal analysis of effects of culture and 5-HTT allele frequency distribution in country of origin.

    Science.gov (United States)

    Kashima, Emiko S; Kent, Stephen; Kashima, Yoshihisa

    2015-01-01

    Life satisfaction of migrants to Australia from 17 countries, assessed at 4-5 months, 16-17 months and 3½ years after arrival, was analyzed with a longitudinal, multilevel analysis. The results indicated that migrants were more satisfied, if the national average life satisfaction was higher in their country of origin, after adjustment for individual-level income, age, and sex and a linear temporal trend. Simultaneously, the migrants were also happier if people in their country of origin had a higher frequency of 5-HTT long allele, a genotype known to be associated with resilience under life stresses. These two relationships were independent, suggesting that both culture and gene matter in international transitions.

  17. The P42 peptide and Peptide-based therapies for Huntington's disease.

    Science.gov (United States)

    Marelli, Cecilia; Maschat, Florence

    2016-03-17

    Huntington's disease (HD) is a progressive neurodegenerative hereditary disease clinically characterised by the presence of involuntary movements, behavioural problems and cognitive decline. The disease-onset is usually between 30 and 50 years of age. HD is a rare disorder affecting approximately 1.3 in 10,000 people in the European Union. It is caused by an expanded CAG repeat in the first exon of the Huntingtin (HTT) gene, leading to an abnormal form of the Huntingtin protein (Htt) (polyQHtt), containing N-terminus, enlarged polyglutamine strands of variable length that stick together to form aggregates and nuclear inclusions in the damaged brain cells. Treatments currently used for Huntington's disease are symptomatic and aimed at temporally relieving the symptoms of the disease; although some promising therapies are on study, there is no drug capable of stopping disease progression either in the form of delaying onset or slowing disability progression. The utilization of peptides interacting with polyQ stretches or with Htt protein to prevent misfolding and aggregation of the expanded polyQ protein is a fascinating idea, because of low potential toxicity and ability to target very initial steps in the pathophysiological cascade of the disease, such as aggregation or cleavage process. Indeed, several therapeutic peptides have been developed and were found to significantly slow down the progression of symptoms in experimental models of Huntington's disease. This review is essentially focusing on the latest development concerning peptide strategy. In particular, we focused on a 23aa peptide P42, which is a part of the Htt protein. It is expected to work principally by preventing the abnormal Htt protein from sticking together, thereby preventing pathological consequences of aggregation and improving the symptoms of the disease. In the meantime, as P42 is part of the Htt protein, some therapeutic properties might be linked to the physiological actions of the

  18. Reciprocal expression of the endocytic protein HIP1R and its repressor FOXP1 predicts outcome in R-CHOP-treated diffuse large B-cell lymphoma patients

    DEFF Research Database (Denmark)

    Wong, K K; Gascoyne, D M; Brown, P J

    2014-01-01

    We previously identified autoantibodies to the endocytic-associated protein Huntingtin-interacting protein 1-related (HIP1R) in diffuse large B-cell lymphoma (DLBCL) patients. HIP1R regulates internalization of cell surface receptors via endocytosis, a process relevant to many therapeutic strateg...

  19. Dominant effects of the Huntington's disease HTT CAG repeat length are captured in gene-expression data sets by a continuous analysis mathematical modeling strategy.

    Science.gov (United States)

    Lee, Jong-Min; Galkina, Ekaterina I; Levantovsky, Rachel M; Fossale, Elisa; Anne Anderson, Mary; Gillis, Tammy; Srinidhi Mysore, Jayalakshmi; Coser, Kathryn R; Shioda, Toshi; Zhang, Bin; Furia, Matthew D; Derry, Jonathan; Kohane, Isaac S; Seong, Ihn Sik; Wheeler, Vanessa C; Gusella, James F; MacDonald, Marcy E

    2013-08-15

    In Huntington's disease (HD), the size of the expanded HTT CAG repeat mutation is the primary driver of the processes that determine age at onset of motor symptoms. However, correlation of cellular biochemical parameters also extends across the normal repeat range, supporting the view that the CAG repeat represents a functional polymorphism with dominant effects determined by the longer allele. A central challenge to defining the functional consequences of this single polymorphism is the difficulty of distinguishing its subtle effects from the multitude of other sources of biological variation. We demonstrate that an analytical approach based upon continuous correlation with CAG size was able to capture the modest (∼21%) contribution of the repeat to the variation in genome-wide gene expression in 107 lymphoblastoid cell lines, with alleles ranging from 15 to 92 CAGs. Furthermore, a mathematical model from an iterative strategy yielded predicted CAG repeat lengths that were significantly positively correlated with true CAG allele size and negatively correlated with age at onset of motor symptoms. Genes negatively correlated with repeat size were also enriched in a set of genes whose expression were CAG-correlated in human HD cerebellum. These findings both reveal the relatively small, but detectable impact of variation in the CAG allele in global data in these peripheral cells and provide a strategy for building multi-dimensional data-driven models of the biological network that drives the HD disease process by continuous analysis across allelic panels of neuronal cells vulnerable to the dominant effects of the HTT CAG repeat.

  20. Multiple aspects of gene dysregulation in Huntington’s Disease.

    Directory of Open Access Journals (Sweden)

    Lara eMoumne

    2013-10-01

    Full Text Available Huntington’s Disease (HD is a genetic neurodegenerative disease caused by a CAG expansion in the gene encoding Huntingtin (Htt. It is characterized by chorea, cognitive and psychiatric disorders. The most affected brain region is the striatum, and the clinical symptoms are directly correlated to the rate of striatal degeneration. The wild-type Htt is a ubiquitous protein and its deletion is lethal. Mutated (expanded Htt produces excitotoxicity, mitochondrial dysfunctions, axonal transport deficit, altered proteasome activity, and gene dysregulation. Transcriptional dysregulation occurs at early neuropathological stages in HD patients. Multiple genes are dysregulated, with overlaps of altered transcripts between mouse models of HD and patient brains. Nuclear localization of Exp-Htt interferes with transcription factors, co-activators and proteins of the transcriptional machinery. Another key mechanism described so far, is an alteration of cytoplasmic retention of the transcriptional repressor REST, which is normally associated with wild-type Htt. As such, Exp-Htt causes alteration of transcription of multiple genes involved in neuronal survival, plasticity, signaling and mitochondrial biogenesis and respiration. Besides these transcriptional dysregulations, Exp-Htt affects the chromatin structure through altered post-translational modifications (PTM of histones and methylation of DNA. Multiple alterations of histone PTM are described, including acetylation, methylation, ubiquitylation, polyamination and phosphorylation. Exp-Htt also affects the expression and regulation of non-coding microRNAs. First multiple neural microRNAs are controlled by REST, and dysregulated in HD, with concomitant de-repression of downstream mRNA targets. Second, Exp-Htt protein or RNA may also play a major role in the processing of miRNAs and hence pathogenesis. These pleiotropic effects of Exp-Htt on gene expression may represent seminal deleterious effects on the

  1. Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington's disease.

    Science.gov (United States)

    Liu, Yanying; Hettinger, Casey L; Zhang, Dong; Rezvani, Khosrow; Wang, Xuejun; Wang, Hongmin

    2014-05-01

    The ubiquitin proteasome system (UPS) is impaired in Huntington's disease, a devastating neurodegenerative disorder. Sulforaphane, a naturally occurring compound, has been shown to stimulate UPS activity in cell cultures. To test whether sulforaphane enhances UPS function in vivo, we treated UPS function reporter mice ubiquitously expressing the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation in the conditions of a healthy UPS. The modified GFP is termed GFP UPS reporter (GFPu). We found that both GFPu and ubiquitinated protein levels were significantly reduced and the three peptidase activities of the proteasome were increased in the brain and peripheral tissues of the mice. Interestingly, sulforaphane treatment also enhanced autophagy activity in the brain and the liver. To further examine whether sulforaphane promotes mutant huntingtin (mHtt) degradation, we treated Huntington's disease cells with sulforaphane and found that sulforaphane not only enhanced mHtt degradation but also reduced mHtt cytotoxicity. Sulforaphane-mediated mHtt degradation was mainly through the UPS pathway as the presence of a proteasome inhibitor abolished this effect. Taken together, these data indicate that sulforaphane activates protein degradation machineries in both the brain and peripheral tissues and may be a therapeutic reagent for Huntington's disease and other intractable disorders. Accumulation of mutant huntingtin (mHtt) protein causes Huntington's disease (HD). Sulforaphane (SFN), a naturally occurring compound, increased proteasome and autophagy activities in vivo and enhanced mHtt turnover and cell survival in HD cell models. SFN-mediated mHtt degradation is mainly through the proteasome pathway. These data suggest that SFN can be a therapeutic reagent for treating HD and other intractable disorders. © 2014 International Society for Neurochemistry.

  2. Clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy": the influence of gender and genetics (CYP2D6, COMT, 5-HTT.

    Directory of Open Access Journals (Sweden)

    Ricardo Pardo-Lozano

    Full Text Available The synthetic psychostimulant MDMA (± 3,4-methylenedioxymethamphetamine, ecstasy acts as an indirect serotonin, dopamine, and norepinephrine agonist and as a mechanism-based inhibitor of the cytochrome P-450 2D6 (CYP2D6. It has been suggested that women are more sensitive to MDMA effects than men but no clinical experimental studies have satisfactorily evaluated the factors contributing to such observations. There are no studies evaluating the influence of genetic polymorphism on the pharmacokinetics (CYP2D6; catechol-O-methyltransferase, COMT and pharmacological effects of MDMA (serotonin transporter, 5-HTT; COMT. This clinical study was designed to evaluate the pharmacokinetics and physiological and subjective effects of MDMA considering gender and the genetic polymorphisms of CYP2D6, COMT, and 5-HTT. A total of 27 (12 women healthy, recreational users of ecstasy were included (all extensive metabolizers for CYP2D6. A single oral weight-adjusted dose of MDMA was administered (1.4 mg/kg, range 75-100 mg which was similar to recreational doses. None of the women were taking oral contraceptives and the experimental session was performed during the early follicular phase of their menstrual cycle. Principal findings show that subjects reached similar MDMA plasma concentrations, and experienced similar positive effects, irrespective of gender or CYP2D6 (not taking into consideration poor or ultra-rapid metabolizers or COMT genotypes. However, HMMA plasma concentrations were linked to CYP2D6 genotype (higher with two functional alleles. Female subjects displayed more intense physiological (heart rate, and oral temperature and negative effects (dizziness, sedation, depression, and psychotic symptoms. Genotypes of COMT val158met or 5-HTTLPR with high functionality (val/val or l/* determined greater cardiovascular effects, and with low functionality (met/* or s/s negative subjective effects (dizziness, anxiety, sedation. In conclusion, the contribution

  3. Influence of 5-HTT variation, childhood trauma and self-efficacy on anxiety traits: a gene-environment-coping interaction study.

    Science.gov (United States)

    Schiele, Miriam A; Ziegler, Christiane; Holitschke, Karoline; Schartner, Christoph; Schmidt, Brigitte; Weber, Heike; Reif, Andreas; Romanos, Marcel; Pauli, Paul; Zwanzger, Peter; Deckert, Jürgen; Domschke, Katharina

    2016-08-01

    Environmental vulnerability factors such as adverse childhood experiences in interaction with genetic risk variants, e.g., the serotonin transporter gene linked polymorphic region (5-HTTLPR), are assumed to play a role in the development of anxiety and affective disorders. However, positive influences such as general self-efficacy (GSE) may exert a compensatory effect on genetic disposition, environmental adversity, and anxiety traits. We, thus, assessed childhood trauma (Childhood Trauma Questionnaire, CTQ) and GSE in 678 adults genotyped for 5-HTTLPR/rs25531 and their interaction on agoraphobic cognitions (Agoraphobic Cognitions Questionnaire, ACQ), social anxiety (Liebowitz Social Anxiety Scale, LSAS), and trait anxiety (State-Trait Anxiety Inventory, STAI-T). The relationship between anxiety traits and childhood trauma was moderated by self-efficacy in 5-HTTLPR/rs25531 LALA genotype carriers: LALA probands maltreated as children showed high anxiety scores when self-efficacy was low, but low anxiety scores in the presence of high self-efficacy despite childhood maltreatment. Our results extend previous findings regarding anxiety-related traits showing an interactive relationship between 5-HTT genotype and adverse childhood experiences by suggesting coping-related measures to function as an additional dimension buffering the effects of a gene-environment risk constellation. Given that anxiety disorders manifest already early in childhood, this insight could contribute to the improvement of psychotherapeutic interventions by including measures strengthening self-efficacy and inform early targeted preventive interventions in at-risk populations, particularly within the crucial time window of childhood and adolescence.

  4. Immobility responses between mouse strains correlate with distinct hippocampal serotonin transporter protein expression and function.

    Science.gov (United States)

    Tang, Man; He, Tao; Meng, Qing-yan; Broussard, John Isaac; Yao, Lan; Diao, Yao; Sang, Xiu-bo; Liu, Qing-peng; Liao, Ying-jun; Li, Yuge; Zhao, Shulei

    2014-11-01

    Mouse strain differences in immobility and in sensitivity to antidepressants have been observed in the forced swimming test (FST) and the tail suspension test (TST). However, the neurotransmitter systems and neural substrates that contribute to these differences remain unknown. To investigate the role of the hippocampal serotonin transporter (5-HTT), we measured baseline immobility and the immobility responses to fluoxetine (FLX) in the FST and the TST in male CD-1, C57BL/6, DBA and BALB/c mice. We observed strain differences in baseline immobility time, with CD-1 mice showing the longest and DBA mice showing the shortest. In contrast, DBA and BALB/c mice showed the highest sensitivity to FLX, whereas CD-1 and C57BL/6 mice showed the lowest sensitivity. Also we found strain differences in both the total 5-HTT protein level and the membrane-bound 5-HTT level (estimated by V max) as follows: DBA>BALB/c>CD-1=C57BL/6. The uptake efficiency of the membrane-bound 5-HTT (estimated by 1/K m) was highest in DBA and BALB/c mice and lowest in CD-1 and C57BL/6 mice. A correlation analysis of subregions within the hippocampus revealed that immobility time was negatively correlated with V max and positively correlated with K m in the hippocampus. Therefore a higher uptake capacity of the membrane-bound 5-HTT in the hippocampus was associated with lower baseline immobility and greater sensitivity to FLX. These results suggest that alterations in hippocampal 5-HTT activity may contribute to mouse strain differences in the FST and the TST.

  5. When cytokinin, a plant hormone, meets the adenosine A2A receptor: a novel neuroprotectant and lead for treating neurodegenerative disorders?

    Directory of Open Access Journals (Sweden)

    Yi-Chao Lee

    Full Text Available It is well known that cytokinins are a class of phytohormones that promote cell division in plant roots and shoots. However, their targets, biological functions, and implications in mammalian systems have rarely been examined. In this study, we show that one cytokinin, zeatin riboside, can prevent pheochromocytoma (PC12 cells from serum deprivation-induced apoptosis by acting on the adenosine A(2A receptor (A(2A-R, which was blocked by an A(2A-R antagonist and a protein kinase A (PKA inhibitor, demonstrating the functional ability of zeatin riboside by mediating through A(2A-R signaling event. Since the A(2A-R was implicated as a therapeutic target in treating Huntington's disease (HD, a cellular model of HD was applied by transfecting mutant huntingtin in PC12 cells. By using filter retardation assay and confocal microscopy we found that zeatin riboside reversed mutant huntingtin (Htt-induced protein aggregations and proteasome deactivation through A(2A-R signaling. PKA inhibitor blocked zeatin riboside-induced suppression of mutant Htt aggregations. In addition, PKA activated proteasome activity and reduced mutant Htt protein aggregations. However, a proteasome inhibitor blocked both zeatin riboside-and PKA activator-mediated suppression of mutant Htt aggregations, confirming mediation of the A(2A-R/PKA/proteasome pathway. Taken together, zeatin riboside might have therapeutic potential as a novel neuroprotectant and a lead for treating neurodegenerative disorders.

  6. An interaction between a polymorphism of the serotonin transporter (5HTT gene and the clinical picture of adolescents with combined type of ADHD (hyperkinetic disorder and youth drinking

    Directory of Open Access Journals (Sweden)

    Gorzkowska, Izabela

    2014-06-01

    Full Text Available Introduction: The combined type of ADHD and alcohol dependence are two different disorders. Research demonstrate that 45-55% of patients diagnosed with ADHD also suffer from comorbid substance abuse, and 11-55% of patients diagnosed with substance abuse suffer from undiagnosed ADHD. Alcohol is by far the most widely used psychoactive substance in the European culture. The serotonin transporter (5HHT gene has been implicated as one of the candidate genes in both disorders in recent molecular genetic research. Aim: The aim of the present study was to seek a common clinical and biological marker for hyperkinetic disorder and youth drinking. Methods: The study was conducted between 2008 and 2012. The sample consisted of 100 combined type ADHD patients: 51 adolescents youth drinking and 100 individuals without mental disorders or addiction in a population-based group. The 5HHT gene polymorphism was examined using PCR (Polymerase Chain Reaction. Statistical analysis was conducted with STATISTICA.PL software (version 5.0.97 licensed by StatSoft, Inc. USA. Results: A preferential trend for the “s” short allele of the investigated 5HHT gene polymorphism was observed in all the groups of adolescents compared to the population-based group of adults without alcohol dependence (p=0.01. Conclusion: Based on the conducted study a provisional conclusion may be drawn that the presence of the short “s” allele of the 5HTT gene polymorphism may be a prognostic factor of impulsivity in ADHD and of predisposition to alcohol dependence.

  7. Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington's disease.

    Science.gov (United States)

    Paul, Bindu D; Sbodio, Juan I; Xu, Risheng; Vandiver, M Scott; Cha, Jiyoung Y; Snowman, Adele M; Snyder, Solomon H

    2014-05-01

    Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.

  8. 改善氧化应激平衡和逆转NET及5-HTT表达异常涉及文拉法辛的抗抑郁作用%Improving oxidative stress response and reversing abnormal expression of hippocampus NET and 5-HTT involved in anti-depressive effect of venlafaxine

    Institute of Scientific and Technical Information of China (English)

    胡小娅; 费慧芝; 蒋心惠; 刘丹; 邱红梅; 周岐新

    2014-01-01

    establish rat depression model. The force swimming test was used to evaluate the behavior chan-ges of experimental rats. The malondialdehyde ( MDA) level and activity of superoxide dismutase ( SOD ) in serum were determined by biochemical methods. The mRNA and protein expressions of NET and 5-HTT in hippocampus were determined by Real-Time Reverse transcription polymerase chain reaction ( real-time RT-PCR) and Western blot ( WB) , respectively. Results Compared with NG rats, obviously increasing immo-bile time of rats in force swimming test and serum MDA level, as well as significantly decreasing SOD activity in serum was observed with clearly decreasing 5-HTT expression and elevating NET expression in hippocam-pus of MG rats. The treatment of venlafaxine distinctly suppressed changes above from CUS-induced rats. However, significant changes failed to be found in VNG rats. Conclusion The anti-depressive effect of venlafaxine may at least partly involve in improving ox-idative stress/anti-oxidative stress balance and revers-ing abnormal expression of NET and 5-HTT.

  9. Mood disorders in Huntington's disease: from behavior to cellular and molecular mechanisms.

    Science.gov (United States)

    Pla, Patrick; Orvoen, Sophie; Saudou, Frédéric; David, Denis J; Humbert, Sandrine

    2014-01-01

    Huntington's disease (HD) is a neurodegenerative disorder that is best known for its effect on motor control. Mood disturbances such as depression, anxiety, and irritability also have a high prevalence in patients with HD, and often start before the onset of motor symptoms. Various rodent models of HD recapitulate the anxiety/depressive behavior seen in patients. HD is caused by an expanded polyglutamine stretch in the N-terminal part of a 350 kDa protein called huntingtin (HTT). HTT is ubiquitously expressed and is implicated in several cellular functions including control of transcription, vesicular trafficking, ciliogenesis, and mitosis. This review summarizes progress in efforts to understand the cellular and molecular mechanisms underlying behavioral disorders in patients with HD. Dysfunctional HTT affects cellular pathways that are involved in mood disorders or in the response to antidepressants, including BDNF/TrkB and serotonergic signaling. Moreover, HTT affects adult hippocampal neurogenesis, a physiological phenomenon that is implicated in some of the behavioral effects of antidepressants and is linked to the control of anxiety. These findings are consistent with the emerging role of wild-type HTT as a crucial component of neuronal development and physiology. Thus, the pathogenic polyQ expansion in HTT could lead to mood disorders not only by the gain of a new toxic function but also by the perturbation of its normal function.

  10. Mood disorders in Huntington’s disease: from behavior to cellular and molecular mechanisms

    Directory of Open Access Journals (Sweden)

    Patrick ePla

    2014-04-01

    Full Text Available Huntington’s disease (HD is a neurodegenerative disorder that is best known for its effect on motor control. Mood disturbances such as depression, anxiety, and irritability also have a high prevalence in patients with HD, and often start before the onset of motor symptoms. Various rodent models of HD recapitulate the anxiety/depressive behavior seen in patients. HD is caused by an expanded polyglutamine stretch in the N-terminal part of a 350 kDa protein called huntingtin (HTT. HTT is ubiquitously expressed and is implicated in several cellular functions including control of transcription, vesicular trafficking, ciliogenesis, and mitosis. This review summarizes progress in efforts to understand the cellular and molecular mechanisms underlying behavioral disorders in patients with HD. Dysfunctional HTT affects cellular pathways that are involved in mood disorders or in the response to antidepressants, including BDNF/TrkB and serotonergic signaling. Moreover, HTT affects adult hippocampal neurogenesis, a physiological phenomenon that is implicated in some of the behavioral effects of antidepressants and is linked to the control of anxiety. These findings are consistent with the emerging role of wild-type HTT as a crucial component of neuronal development and physiology. Thus, the pathogenic polyQ expansion in HTT could lead to mood disorders not only by the gain of a new toxic function but also by the perturbation of its normal function.

  11. Amitriptyline improves motor function via enhanced neurotrophin signaling and mitochondrial functions in the murine N171-82Q Huntington disease model.

    Science.gov (United States)

    Cong, Wei-Na; Chadwick, Wayne; Wang, Rui; Daimon, Caitlin M; Cai, Huan; Amma, Jennifer; Wood, William H; Becker, Kevin G; Martin, Bronwen; Maudsley, Stuart

    2015-01-30

    Huntington disease (HD) is a neurodegenerative disorder characterized by progressive motor impairment and cognitive alterations. Hereditary HD is primarily caused by the expansion of a CAG trinucleotide repeat in the huntingtin (Htt) gene, which results in the production of mutant huntingtin protein (mHTT) with an expanded amino-terminal polyglutamine (poly(Q)) stretch. Besides pathological mHTT aggregation, reduced brain-derived neurotrophic factor (BDNF) levels, impaired neurotrophin signaling, and compromised mitochondrial functions also contribute to the deleterious progressive etiology of HD. As a well tolerated Food and Drug Administration-approved antidepressant, amitriptyline (AMI) has shown efficacy in treating neurodegenerative murine models via potentiation of BDNF levels and amelioration of alterations in neurotrophin signaling pathways. In this study, we observed profound improvements in the motor coordination of AMI-treated N171-82Q HD model mice. The beneficial effects of AMI treatment were associated with its ability to reduce mHTT aggregation, potentiation of the BDNF-TrkB signaling system, and support of mitochondrial integrity and functionality. Our study not only provides preclinical evidence for the therapeutic potency of AMI in treating HD, but it also represents an important example of the usefulness of additional pharmacogenomic profiling of pre-existing drugs for novel therapeutic effects with often intractable pathological scenarios. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Systematic analysis of fly models with multiple drivers reveals different effects of ataxin-1 and huntingtin in neuron subtype-specific expression.

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    Risa Shiraishi

    Full Text Available The fruit fly, Drosophila melanogaster, is a commonly used model organism for neurodegenerative diseases. Its major advantages include a short lifespan and its susceptibility to manipulation using sophisticated genetic techniques. Here, we report the systematic comparison of fly models of two polyglutamine (polyQ diseases. We induced expression of the normal and mutant forms of full-length Ataxin-1 and Huntingtin exon 1 in cholinergic, dopaminergic, and motor neurons, and glial cells using cell type-specific drivers. We systematically analyzed their effects based on multiple phenotypes: eclosion rate, lifespan, motor performance, and circadian rhythms of spontaneous activity. This systematic assay system enabled us to quantitatively evaluate and compare the functional disabilities of different genotypes. The results suggest different effects of Ataxin-1 and Huntingtin on specific types of neural cells during development and in adulthood. In addition, we confirmed the therapeutic effects of LiCl and butyrate using representative models. These results support the usefulness of this assay system for screening candidate chemical compounds that modify the pathologies of polyQ diseases.

  13. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  14. Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death.

    Science.gov (United States)

    Parker, J A; Connolly, J B; Wellington, C; Hayden, M; Dausset, J; Neri, C

    2001-11-06

    Huntington's disease (HD) is a dominant neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). HD pathogenesis appears to involve the production of mutated N-terminal htt, cytoplasmic and nuclear aggregation of htt, and abnormal activity of htt interactor proteins essential to neuronal survival. Before cell death, neuronal dysfunction may be an important step of HD pathogenesis. To explore polyQ-mediated neuronal toxicity, we expressed the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins in the six touch receptor neurons of Caenorhabditis elegans. Expanded polyQ produced touch insensitivity in young adults. Noticeably, only 28 +/- 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform in vivo suppression of polyQ-mediated neuronal dysfunction.

  15. Ca(2+) handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease.

    Science.gov (United States)

    Pellman, Jessica J; Hamilton, James; Brustovetsky, Tatiana; Brustovetsky, Nickolay

    2015-08-01

    We investigated Ca(2+) handling in isolated brain synaptic and non-synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non-synaptic mitochondria from 2- and 12-month-old YAC128 mice had larger Ca(2+) uptake capacity than mitochondria from YAC18 and wild-type FVB/NJ mice. Synaptic mitochondria from 12-month-old YAC128 mice had further augmented Ca(2+) capacity compared with mitochondria from 2-month-old YAC128 mice and age-matched YAC18 and FVB/NJ mice. This increase in Ca(2+) uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12-month-old YAC128 mice. We speculate that this may happen because of mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca(2+) followed by recovery to near resting levels. Following recovery of cytosolic Ca(2+), mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca(2+), suggesting similar Ca(2+) release and, consequently, Ca(2+) loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca(2+) handling in brain mitochondria of YAC128 mice. We demonstrate that mutant huntingtin (mHtt) binds to brain synaptic and nonsynaptic mitochondria and the amount of mitochondria-bound mHtt correlates with increased mitochondrial Ca(2+) uptake capacity. We propose that this may happen due to mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage.

  16. A pathogenic mechanism in Huntington's disease involves small CAG-repeated RNAs with neurotoxic activity.

    Science.gov (United States)

    Bañez-Coronel, Mónica; Porta, Silvia; Kagerbauer, Birgit; Mateu-Huertas, Elisabet; Pantano, Lorena; Ferrer, Isidre; Guzmán, Manuel; Estivill, Xavier; Martí, Eulàlia

    2012-01-01

    Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.

  17. Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington’s disease

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    Magdalena eCzeredys

    2013-11-01

    Full Text Available Huntington’s disease (HD is a hereditary neurodegenerative disease caused by the expansion of a polyglutamine stretch in the huntingtin (HTT protein and characterized by dysregulated calcium homeostasis. We investigated whether these disturbances are correlated with changes in the mRNA level of the genes that encode proteins involved in calcium homeostasis and signaling (i.e., the calciosome. Using custom-made TaqMan low-density arrays containing probes for 96 genes, we quantified mRNA in the striatum in YAC128 mice, a model of HD, and wildtype mice. HTT mutation caused the increased expression of some components of the calcium signalosome, including calretinin, presenilin 2, and calmyrin 1, and the increased expression of genes indirectly involved in calcium homeostasis, such as huntingtin-associated protein 1 and calcyclin-binding protein. To verify these findings in a different model, we used PC12 cells with an inducible expression of mutated full-length HTT. Using single-cell imaging with Fura-2AM, we found that store-operated Ca2+ entry but not endoplasmic reticulum store content was changed as a result of the expression of mutant HTT. Statistically significant downregulation of the Orai calcium channel subunit 2, calmodulin, and septin 4 was detected in cells that expressed mutated HTT. Our data indicate that the dysregulation of calcium homeostasis correlates with changes in the gene expression of members of the calciosome. These changes, however, differed in the two models of HD used in this study. Our results indicate that each HD model exhibits distinct features that may only partially resemble the human disease.

  18. Cells exposed to a huntingtin fragment containing an expanded polyglutamine tract show no sign of ion channel formation: results arguing against the ion channel hypothesis

    DEFF Research Database (Denmark)

    Nørremølle, Anne; Grunnet, Morten; Hasholt, Lis

    2003-01-01

    tract to form ion channels in two cell types. Whole cell current from Xenopus oocytes was recorded using two-electrode voltage-clamp technique, and whole cell current from CHO-K1 cells was recorded by patch-clamp technique. The fragment with an expanded polyglutamine sequence induced no change......Ion channels formed by expanded polyglutamine tracts have been proposed to play an important role in the pathological processes leading to neurodegeneration in Huntington's disease and other CAG repeat diseases. We tested the capacity of a huntingtin fragment containing an expanded polyglutamine...... in the currents recorded in any of the two expression systems, indicating no changes in ion channel activity. The results therefore argue against the proposed hypothesis of expanded polyglutamines forming ion channels....

  19. Intravitreal administration of HA-1077, a ROCK inhibitor, improves retinal function in a mouse model of huntington disease.

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    Mei Li

    Full Text Available Huntington disease (HD is an inherited neurodegenerative disease that affects multiple brain regions. It is caused by an expanded polyglutamine tract in huntingtin (Htt. The development of therapies for HD and other neurodegenerative diseases has been hampered by multiple factors, including the lack of clear therapeutic targets, and the cost and complexity of testing lead compounds in vivo. The R6/2 HD mouse model is widely used for pre-clinical trials because of its progressive and robust neural dysfunction, which includes retinal degeneration. Profilin-1 is a Htt binding protein that inhibits Htt aggregation. Its binding to Htt is regulated by the rho-associated kinase (ROCK, which phosphorylates profilin at Ser-137. ROCK is thus a therapeutic target in HD. The ROCK inhibitor Y-27632 reduces Htt toxicity in fly and mouse models. Here we characterized the progressive retinopathy of R6/2 mice between 6-19 weeks of age to determine an optimal treatment window. We then tested a clinically approved ROCK inhibitor, HA-1077, administered intravitreally via liposome-mediated drug delivery. HA-1077 increased photopic and flicker ERG response amplitudes in R6/2 mice, but not in wild-type littermate controls. By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.

  20. Quantitative Electroencephalographic Biomarkers in Preclinical and Human Studies of Huntington's Disease: Are They Fit-for-Purpose for Treatment Development?

    Science.gov (United States)

    Leuchter, Michael K; Donzis, Elissa J; Cepeda, Carlos; Hunter, Aimee M; Estrada-Sánchez, Ana María; Cook, Ian A; Levine, Michael S; Leuchter, Andrew F

    2017-01-01

    A major focus in development of novel therapies for Huntington's disease (HD) is identification of treatments that reduce the burden of mutant huntingtin (mHTT) protein in the brain. In order to identify and test the efficacy of such therapies, it is essential to have biomarkers that are sensitive to the effects of mHTT on brain function to determine whether the intervention has been effective at preventing toxicity in target brain systems before onset of clinical symptoms. Ideally, such biomarkers should have a plausible physiologic basis for detecting the effects of mHTT, be measureable both in preclinical models and human studies, be practical to measure serially in clinical trials, and be reliably measurable in HD gene expansion carriers (HDGECs), among other features. Quantitative electroencephalography (qEEG) fulfills many of these basic criteria of a "fit-for-purpose" biomarker. qEEG measures brain oscillatory activity that is regulated by the brain structures that are affected by mHTT in premanifest and early symptom individuals. The technology is practical to implement in the laboratory and is well tolerated by humans in clinical trials. The biomarkers are measureable across animal models and humans, with findings that appear to be detectable in HDGECs and translate across species. We review here the literature on recent developments in both preclinical and human studies of the use of qEEG biomarkers in HD, and the evidence for their usefulness as biomarkers to help guide development of novel mHTT lowering treatments.

  1. Advances in huntington disease drug discovery: novel approaches to model disease phenotypes.

    Science.gov (United States)

    Bard, Jonathan; Wall, Michael D; Lazari, Ovadia; Arjomand, Jamshid; Munoz-Sanjuan, Ignacio

    2014-02-01

    Huntington disease is a monogenic, autosomal dominant, progressive neurodegenerative disorder caused by a trinucleotide CAG repeat expansion in exon 1 of the huntingtin (HTT) gene; age of onset of clinical symptoms inversely correlates with expanded CAG repeat length. HD leads to extensive degeneration of the basal ganglia, hypothalamic nuclei, and selected cortical areas, and a wide range of molecular mechanisms have been implicated in disease pathology in animal or cellular models expressing mutated HTT (mHTT) proteins, either full-length or amino-terminal fragments. However, HD cellular models that recapitulate the slow progression of the disease have not been available due to the toxicity of overexpressed exogenous mHTT or to limitations with using primary cells for long-term studies. Most investigations of the effects of mHTT relied on cytotoxicity or aggregation end points in heterologous systems or in primary embryonic neuroglial cultures derived from HD mouse models. More innovative approaches are currently under active investigation, including screening using electrophysiological endpoints, as well as the recent use of primary blood mononuclear cells and of human embryonic stem cells derived from a variety of HD research participants. Here we describe how these cellular systems are being used to investigate HD biology as well as to identify mechanisms with therapeutic potential.

  2. Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates.

    Directory of Open Access Journals (Sweden)

    Maggie P Wear

    Full Text Available Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12 cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID domains (≥ 100 amino acids and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.

  3. Short G-rich oligonucleotides as a potential therapeutic for Huntington's Disease

    Directory of Open Access Journals (Sweden)

    Parekh-Olmedo Hetal

    2006-10-01

    Full Text Available Abstract Background Huntington's Disease (HD is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt is produced by HD genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions. Results As such, efforts to find a therapy for HD have focused on agents that disrupt or block the mutant Htt aggregation pathway. Here, we report that short guanosine monotonic oligonucleotides capable of adopting a G-quartet structure, are effective inhibitors of aggregation. By utilizing a biochemical/immunoblotting assay as an initial screen, we identified a 20-mer, all G-oligonucleotide (HDG as an active molecule. Subsequent testing in a cell-based assay revealed that HDG was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant Htt fragment and green fluorescent protein (eGFP. Taken together, our results suggest that a monotonic G-oligonucleotide, capable of adopting a G-quartet conformation is an effective inhibitor of aggregation. This oligonucleotide can also enable cell survival in PC12 cells overexpressing a mutant Htt fragment fusion gene. Conclusion Single-stranded DNA oligonucleotides capable of forming stable G-quartets can inhibit aggregation of the mutant Htt fragment protein. This activity maybe an important part of the pathogenecity of Huntington's Disease. Our results reveal a new class of agents that could be developed as a therapeutic approach for Huntington's Disease.

  4. Correlations of behavioral deficits with brain pathology assessed through longitudinal MRI and histopathology in the R6/1 mouse model of Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Ivan Rattray

    Full Text Available Huntington's disease (HD is caused by the expansion of a CAG repeat in the huntingtin (HTT gene. The R6 mouse models of HD express a mutant version of exon 1 HTT and typically develop motor and cognitive impairments, a widespread huntingtin (HTT aggregate pathology and brain atrophy. Unlike the more commonly used R6/2 mouse line, R6/1 mice have fewer CAG repeats and, subsequently, a less rapid pathological decline. Compared to the R6/2 line, fewer descriptions of the progressive pathologies exhibited by R6/1 mice exist. The association between the molecular and cellular neuropathology with brain atrophy, and with the development of behavioral phenotypes remains poorly understood in many models of HD. In attempt to link these factors in the R6/1 mouse line, we have performed detailed assessments of behavior and of regional brain abnormalities determined through longitudinal, in vivo magnetic resonance imaging (MRI, as well as an end-stage, ex vivo MRI study and histological assessment. We found progressive decline in both motor and non-motor related behavioral tasks in R6/1 mice, first evident at 11 weeks of age. Regional brain volumes were generally unaffected at 9 weeks, but by 17 weeks there was significant grey matter atrophy. This age-related brain volume loss was validated using a more precise, semi-automated Tensor Based morphometry assessment. As well as these clear progressive phenotypes, mutant HTT (mHTT protein, the hallmark of HD molecular pathology, was widely distributed throughout the R6/1 brain and was accompanied by neuronal loss. Despite these seemingly concomitant, robust pathological phenotypes, there appeared to be little correlation between the three main outcome measures: behavioral performance, MRI-detected brain atrophy and histopathology. In conclusion, R6/1 mice exhibit many features of HD, but the underlying mechanisms driving these clear behavioral disturbances and the brain volume loss, still remain unclear.

  5. Correlations of behavioral deficits with brain pathology assessed through longitudinal MRI and histopathology in the R6/2 mouse model of HD.

    Directory of Open Access Journals (Sweden)

    Ivan Rattray

    Full Text Available Huntington's disease (HD is caused by the expansion of a CAG repeat in the huntingtin (HTT gene. The R6/2 mouse model of HD expresses a mutant version of exon 1 HTT and develops motor and cognitive impairments, a widespread huntingtin (HTT aggregate pathology and brain atrophy. Despite the vast number of studies that have been performed on this model, the association between the molecular and cellular neuropathology with brain atrophy, and with the development of behavioral phenotypes remains poorly understood. In an attempt to link these factors, we have performed longitudinal assessments of behavior (rotarod, open field, passive avoidance and of regional brain abnormalities determined through magnetic resonance imaging (MRI (whole brain, striatum, cortex, hippocampus, corpus callosum, as well as an end-stage histological assessment. Detailed correlative analyses of these three measures were then performed. We found a gender-dependent emergence of motor impairments that was associated with an age-related loss of regional brain volumes. MRI measurements further indicated that there was no striatal atrophy, but rather a lack of striatal growth beyond 8 weeks of age. T2 relaxivity further indicated tissue-level changes within brain regions. Despite these dramatic motor and neuroanatomical abnormalities, R6/2 mice did not exhibit neuronal loss in the striatum or motor cortex, although there was a significant increase in neuronal density due to tissue atrophy. The deposition of the mutant HTT (mHTT protein, the hallmark of HD molecular pathology, was widely distributed throughout the brain. End-stage histopathological assessments were not found to be as robustly correlated with the longitudinal measures of brain atrophy or motor impairments. In conclusion, modeling pre-manifest and early progression of the disease in more slowly progressing animal models will be key to establishing which changes are causally related.

  6. Striatal Vulnerability in Huntington’s Disease: Neuroprotection Versus Neurotoxicity

    Science.gov (United States)

    Morigaki, Ryoma; Goto, Satoshi

    2017-01-01

    Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat encoding an abnormally long polyglutamine tract (PolyQ) in the huntingtin (Htt) protein. In HD, striking neuropathological changes occur in the striatum, including loss of medium spiny neurons and parvalbumin-expressing interneurons accompanied by neurodegeneration of the striosome and matrix compartments, leading to progressive impairment of reasoning, walking and speaking abilities. The precise cause of striatal pathology in HD is still unknown; however, accumulating clinical and experimental evidence suggests multiple plausible pathophysiological mechanisms underlying striatal neurodegeneration in HD. Here, we review and discuss the characteristic neurodegenerative patterns observed in the striatum of HD patients and consider the role of various huntingtin-related and striatum-enriched proteins in neurotoxicity and neuroprotection. PMID:28590448

  7. Striatal Vulnerability in Huntington's Disease: Neuroprotection Versus Neurotoxicity.

    Science.gov (United States)

    Morigaki, Ryoma; Goto, Satoshi

    2017-06-07

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat encoding an abnormally long polyglutamine tract (PolyQ) in the huntingtin (Htt) protein. In HD, striking neuropathological changes occur in the striatum, including loss of medium spiny neurons and parvalbumin-expressing interneurons accompanied by neurodegeneration of the striosome and matrix compartments, leading to progressive impairment of reasoning, walking and speaking abilities. The precise cause of striatal pathology in HD is still unknown; however, accumulating clinical and experimental evidence suggests multiple plausible pathophysiological mechanisms underlying striatal neurodegeneration in HD. Here, we review and discuss the characteristic neurodegenerative patterns observed in the striatum of HD patients and consider the role of various huntingtin-related and striatum-enriched proteins in neurotoxicity and neuroprotection.

  8. Protein oxidation in Huntington disease.

    Science.gov (United States)

    Sorolla, M Alba; Rodríguez-Colman, María José; Vall-llaura, Núria; Tamarit, Jordi; Ros, Joaquim; Cabiscol, Elisa

    2012-01-01

    Huntington disease (HD) is an inherited neurodegenerative disorder caused by expansion of CAG repeats in the huntingtin gene, affecting initially the striatum and progressively the cortex. Oxidative stress, and consequent protein oxidation, has been described as important to disease progression. This review focuses on recent advances in the field, with a particular emphasis on the identified target proteins and the role that their oxidation has or might have in the pathophysiology of HD. Oxidation and the resulting inactivation and/or degradation of important proteins can explain the impairment of several metabolic pathways in HD. Oxidation of enzymes involved in ATP synthesis can account for the energy deficiency observed. Impairment of protein folding and degradation can be due to oxidation of several heat shock proteins and Valosin-containing protein. Oxidation of two enzymes involved in the vitamin B6 metabolism could result in decreased availability of pyridoxal phosphate, which is a necessary cofactor in transaminations, the kynurenine pathway and the synthesis of glutathione, GABA, dopamine and serotonin, all of which have a key role in HD pathology. In addition, protein oxidation often contributes to oxidative stress, aggravating the molecular damage inside the cell. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  9. Profile of pridopidine and its potential in the treatment of Huntington disease: the evidence to date

    Directory of Open Access Journals (Sweden)

    Squitieri F

    2015-10-01

    Full Text Available Ferdinando Squitieri,1 Justo Garcia de Yebenes2 1IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo and Mendel Institute of Human Genetics, Rome, Italy; 2Fundación para Investigaciones Neurológicas, Madrid, Spain Abstract: Huntington disease (HD is a chronic, genetic, neurodegenerative disease for which there is no cure. The main symptoms of HD are abnormal involuntary movements (chorea and dystonia, impaired voluntary movements (ie, incoordination and gait balance, progressive cognitive decline, and psychiatric disturbances. HD is caused by a CAG-repeat expanded mutation in the HTT gene, which encodes the huntingtin protein. The inherited mutation results in the production of an elongated polyQ mutant huntingtin protein (mHtt. The cellular functions of the Htt protein are not yet fully understood, but the functions of its mutant variant are thought to include alteration of gene transcription and energy production, and dysregulation of neurotransmitter metabolism, receptors, and growth factors. The phenylpiperidines pridopidine (4-[3-methanesulfonyl-phenyl]-1-propyl-piperidine; formerly known as ACR16 and OSU6162 ([S]-[-]-3-[3-methane [sulfonyl-phenyl]-1-propyl-piperidine are members of a new class of pharmacologic agents known as “dopamine stabilizers”. Recent clinical trials have highlighted the potential of pridopidine for symptomatic treatment of patients with HD. More recently, the analysis of HD models (ie, in vitro and in mice highlighted previously unknown effects of pridopidine (increase in brain-derived neurotrophic factor, reduction in mHtt levels, and σ-1 receptor binding and modulation. These additional functions of pridopidine suggest it might be a neuroprotective and disease-modifying drug. Data from ongoing clinical trials of pridopidine will help define its place in the treatment of HD. This commentary examines the available preclinical and clinical evidence regarding the use of pridopidine in HD. Keywords

  10. Profile of pridopidine and its potential in the treatment of Huntington disease: the evidence to date.

    Science.gov (United States)

    Squitieri, Ferdinando; de Yebenes, Justo Garcia

    2015-01-01

    Huntington disease (HD) is a chronic, genetic, neurodegenerative disease for which there is no cure. The main symptoms of HD are abnormal involuntary movements (chorea and dystonia), impaired voluntary movements (ie, incoordination and gait balance), progressive cognitive decline, and psychiatric disturbances. HD is caused by a CAG-repeat expanded mutation in the HTT gene, which encodes the huntingtin protein. The inherited mutation results in the production of an elongated polyQ mutant huntingtin protein (mHtt). The cellular functions of the Htt protein are not yet fully understood, but the functions of its mutant variant are thought to include alteration of gene transcription and energy production, and dysregulation of neurotransmitter metabolism, receptors, and growth factors. The phenylpiperidines pridopidine (4-[3-methanesulfonyl-phenyl]-1-propyl-piperidine; formerly known as ACR16) and OSU6162 ([S]-[-]-3-[3-methane [sulfonyl-phenyl]-1-propyl-piperidine) are members of a new class of pharmacologic agents known as "dopamine stabilizers". Recent clinical trials have highlighted the potential of pridopidine for symptomatic treatment of patients with HD. More recently, the analysis of HD models (ie, in vitro and in mice) highlighted previously unknown effects of pridopidine (increase in brain-derived neurotrophic factor, reduction in mHtt levels, and σ-1 receptor binding and modulation). These additional functions of pridopidine suggest it might be a neuroprotective and disease-modifying drug. Data from ongoing clinical trials of pridopidine will help define its place in the treatment of HD. This commentary examines the available preclinical and clinical evidence regarding the use of pridopidine in HD.

  11. Neuronal Ca(2+) dyshomeostasis in Huntington disease.

    Science.gov (United States)

    Giacomello, Marta; Oliveros, Juan C; Naranjo, Jose R; Carafoli, Ernesto

    2013-01-01

    The expansion of the N-terminal poly-glutamine tract of the huntingtin (Htt) protein is responsible for Huntington disease (HD). A large number of studies have explored the neuronal phenotype of HD, but the molecular aethiology of the disease is still very poorly understood. This has hampered the development of an appropriate therapeutical strategy to at least alleviate its symptoms. In this short review, we have focused our attention on the alteration of a specific cellular mechanism common to all HD models, either genetic or induced by treatment with 3-NPA, i.e. the cellular dyshomeostasis of Ca(2+). We have highlighted the direct and indirect (i.e. transcriptionally mediated) effects of mutated Htt on the maintenance of the intracellular Ca(2+) balance, the correct modulation of which is fundamental to cell survival and the disturbance of which plays a key role in the death of the cell.

  12. Transgenic animal models for study of the pathogenesis of Huntington's disease and therapy.

    Science.gov (United States)

    Chang, Renbao; Liu, Xudong; Li, Shihua; Li, Xiao-Jiang

    2015-01-01

    Huntington's disease (HD) is caused by a genetic mutation that results in polyglutamine expansion in the N-terminal regions of huntingtin. As a result, this polyQ expansion leads to the misfolding and aggregation of mutant huntingtin as well as age-dependent neurodegeneration. The genetic mutation in HD allows for generating a variety of animal models that express different forms of mutant huntingtin and show differential pathology. Studies of these animal models have provided an important insight into the pathogenesis of HD. Mouse models of HD include transgenic mice, which express N-terminal or full-length mutant huntingtin ubiquitously or selectively in different cell types, and knock-in mice that express full-length mutant Htt at the endogenous level. Large animals, such as pig, sheep, and monkeys, have also been used to generate animal HD models. This review focuses on the different features of commonly used transgenic HD mouse models as well as transgenic large animal models of HD, and also discusses how to use them to identify potential therapeutics. Since HD shares many pathological features with other neurodegenerative diseases, identification of therapies for HD would also help to develop effective treatment for different neurodegenerative diseases that are also caused by protein misfolding and occur in an age-dependent manner.

  13. Glutathione redox cycle dysregulation in Huntington’s disease knock-in striatal cells

    OpenAIRE

    Ribeiro, Márcio; Rosenstock, Tatiana; cunha-oliveira, teresa; Ferreira, Ildete; Oliveira, Catarina R.; Rego, Ana Cristina

    2012-01-01

    Huntington’s disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knoc...

  14. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins.

    Science.gov (United States)

    Matsumoto, Gen; Wada, Koji; Okuno, Misako; Kurosawa, Masaru; Nukina, Nobuyuki

    2011-10-21

    Selective macroautophagy (autophagy) of ubiquitinated protein is implicated as a compensatory mechanism of the ubiquitin-proteasome system. p62/SQSTM1 is a key molecule managing autophagic clearance of polyubiquitinated proteins. However, little is known about mechanisms controlling autophagic degradation of polyubiquitinated proteins. Here, we show that the specific phosphorylation of p62 at serine 403 (S403) in its ubiquitin-associated (UBA) domain increases the affinity between UBA and polyubiquitin chain, resulting in efficiently targeting polyubiquitinated proteins in "sequestosomes" and stabilizing sequestosome structure as a cargo of ubiquitinated proteins for autophagosome entry. Casein kinase 2 (CK2) phosphorylates S403 of p62 directly. Furthermore, CK2 overexpression or phosphatase inhibition reduces the formation of inclusion bodies of the polyglutamine-expanded huntingtin exon1 fragment in a p62-dependent manner. We propose that phosphorylation of p62 at S403 regulates autophagic clearance of ubiquitinated proteins and protein aggregates that are poorly degraded by proteasomes.

  15. Of mice, rats and men: Revisiting the quinolinic acid hypothesis of Huntington's disease.

    Science.gov (United States)

    Schwarcz, Robert; Guidetti, Paolo; Sathyasaikumar, Korrapati V; Muchowski, Paul J

    2010-02-09

    The neurodegenerative disease Huntington's disease (HD) is caused by an expanded polyglutamine (polyQ) tract in the protein huntingtin (htt). Although the gene encoding htt was identified and cloned more than 15 years ago, and in spite of impressive efforts to unravel the mechanism(s) by which mutant htt induces nerve cell death, these studies have so far not led to a good understanding of pathophysiology or an effective therapy. Set against a historical background, we review data supporting the idea that metabolites of the kynurenine pathway (KP) of tryptophan degradation provide a critical link between mutant htt and the pathophysiology of HD. New studies in HD brain and genetic model organisms suggest that the disease may in fact be causally related to early abnormalities in KP metabolism, favoring the formation of two neurotoxic metabolites, 3-hydroxykynurenine and quinolinic acid, over the related neuroprotective agent kynurenic acid. These findings not only link the excitotoxic hypothesis of HD pathology to an impairment of the KP but also define new drug targets and therefore have direct therapeutic implications. Thus, pharmacological normalization of the imbalance in brain KP metabolism may provide clinical benefits, which could be especially effective in early stages of the disease.

  16. Antidepressants for neuroprotection in Huntington's disease: A review.

    Science.gov (United States)

    Jamwal, Sumit; Kumar, Puneet

    2015-12-15

    Huntington Disease (HD), which is characterized by abnormal dance-like movements, is a neurodegenerative disorder caused by a genetic mutation that results in an expanded polyglutamine stretch in the NH2 terminus of huntingtin protein (HTT). The principal neuropathological hallmarks of disease include loss of striatal and cortical projection neurons. HTT is ubiquitously expressed and is implicated in several cellular functions including neurogenesis, cell trafficking and brain-derived neurotrophic factor (BDNF) production. Major depression is the most common symptom among pre-symptomatic HD carriers and numerous pieces of preclinical evidence have suggested the use of antidepressants in HD not only elevates mood but also slows down the disease progression by activating different neuroprotective mechanism like BDNF/TrkB pathway, MAPK/ERK signalling, neurogenesis and Wnt signalling. HTT plays major role in neurogenesis, a physiological phenomenon that is implicated in some of the behavioral effects of antidepressants. Currently, there is no clinically available treatment that can halt or slow down the progression of HD except tetrabenazine (the only FDA approved drug); however, this drug also induces depression and sedation in patients. In this review, a brief discussion has been made about the mutant HTT that induced various cellular and molecular mechanisms underlying behavioral disorders in HD. Further, an attempt has been made to understand the various cellular mechanisms involved in mediating the neuroprotective effects of antidepressants in HD. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. The de-ubiquitinating enzyme ataxin-3 does not modulate disease progression in a knock-in mouse model of Huntington disease.

    Science.gov (United States)

    Zeng, Li; Tallaksen-Greene, Sara J; Wang, Bo; Albin, Roger L; Paulson, Henry L

    2013-01-01

    Ataxin-3 is a deubiquitinating enzyme (DUB) that participates in ubiquitin-dependent protein quality control pathways and, based on studies in model systems, may be neuroprotective against toxic polyglutamine proteins such as the Huntington's disease (HD) protein, huntingtin (htt). HD is one of at least nine polyglutamine neurodegenerative diseases in which disease-causing proteins accumulate in ubiquitin-positive inclusions within neurons. In studies crossing mice null for ataxin-3 to an established HD knock-in mouse model (HdhQ200), we tested whether loss of ataxin-3 alters disease progression, perhaps by impairing the clearance of mutant htt or the ubiquitination of inclusions. While loss of ataxin-3 mildly exacerbated age-dependent motor deficits, it did not alter inclusion formation, ubiquitination of inclusions or levels of mutant or normal htt. Ataxin-3, itself a polyglutamine-containing protein with multiple ubiquitin binding domains, was not observed to localize to htt inclusions. Changes in neurotransmitter receptor binding known to occur in HD knock-in mice also were not altered by the loss of ataxin-3, although we unexpectedly observed increased GABAA receptor binding in the striatum of HdhQ200 mice, which has not previously been noted. Finally, we confirmed that CNS levels of hsp70 are decreased in HD mice as has been reported in other HD mouse models, regardless of the presence or absence of ataxin-3. We conclude that while ataxin-3 may participate in protein quality control pathways, it does not critically regulate the handling of mutant htt or contribute to major features of disease pathogenesis in HD.

  18. The value of 5-HTT gene polymorphism for the assessment and prediction of male adolescence violence%5-羟色胺转运体基因多态性与青少年暴力犯罪行为的相关性研究

    Institute of Scientific and Technical Information of China (English)

    于跃; 刘祥; 杨振兴; 邱昌建; 马小红

    2012-01-01

    Objective To establish an adolescent violence crime prediction model,and to assess the value of serotonin transporter (5-HTT)gene polymorphism for the assessment and prediction of violent crime.Methods Investigative tools were used to analyze the difference in personality dimensions,social support,coping styles,aggressiveness,impulsivity,and family condition scale between 223 adolescents with violence behavior and 148 adolescents without violence behavior. The distribution of 5-HTT gene polymorphisms (5-HTTLPR and 5-HTTVNTR) was compared between the two groups.The role of 5-HTT gene polymorphism on adolescent personality,impulsion and aggression scale also was also analyzed.Stepwise logistic regression was used to establish a predictive model for adolescent violent crime.Results Significant difference was found between the violence group and the control group on multiple dimensions of psychology and environment scales.However,no statistical difference was found with regard to the 5-HTT genotypes and alleles between adolescents with violent behaviors and normal controls.The rate of prediction accuracy was not significantly improved when 5-HTT gene polymorphism was taken into the model.Conclusion The violent crime of adolescents was closely related with social and environmental factors.No association was found between 5-HTT polymorphisms and adolescent violence criminal behavior.%目的 通过建立青少年暴力犯罪模型,探讨5-羟色胺转运体(serotonin transporter,5-HTT)基因多态性在暴力犯罪评估预测中的作用.方法 使用调查量表分析223例暴力组和148名对照组青少年在人格、社会支持、应付方式、冲动性、攻击性和家庭情况等方面的差异.分别比较暴力组和正常对照组5-HTT基因多态位点5-HTTVNTR、5-HTTLPR基因型与等位基因的分布,以观察5-HTT基因多态性与青少年人格、冲动和攻击等分量表的关系.采用逐步Logistic回归建立青少年

  19. Extended polyglutamine tracts cause aggregation and structural perturbation of an adjacent beta barrel protein.

    Science.gov (United States)

    Ignatova, Zoya; Gierasch, Lila M

    2006-05-05

    Formation of fibrillar intranuclear inclusions and related neuropathologies of the CAG-repeat disorders are linked to the expansion of a polyglutamine tract. Despite considerable effort, the etiology of these devastating diseases remains unclear. Although polypeptides with glutamine tracts recapitulate many of the observed characteristics of the gene products with CAG repeats, such as in vitro and in vivo aggregation and toxicity in model organisms, extended polyglutamine segments have also been reported to structurally perturb proteins into which they are inserted. Additionally, the sequence context of a polyglutamine tract has recently been shown to modulate its propensity to aggregate. These findings raise the possibility that indirect influences of the repeat tract on adjacent protein domains are contributory to pathologies. Destabilization of an adjacent domain may lead to loss of function, as well as favoring non-native structures in the neighboring domain causing them to be prone to intermolecular association and consequent aggregation. To explore these phenomena, we have used chimeras of a well studied globular protein and exon 1 of huntingtin. We find that expansion of the polyglutamine segment beyond the pathological threshold (>35 glutamines) results in structural perturbation of the neighboring protein whether the huntingtin exon is N- or C-terminal. Elongation of the polyglutamine region also substantially increases the propensity of the chimera to aggregate, both in vitro and in vivo, and in vitro aggregation kinetics of a chimera with a 53-glutamine repeat follow a nucleation polymerization mechanism with a monomeric nucleus.

  20. Integrated regulation of motor-driven organelle transport by scaffolding proteins.

    Science.gov (United States)

    Fu, Meng-meng; Holzbaur, Erika L F

    2014-10-01

    Intracellular trafficking pathways, including endocytosis, autophagy, and secretion, rely on directed organelle transport driven by the opposing microtubule motor proteins kinesin and dynein. Precise spatial and temporal targeting of vesicles and organelles requires the integrated regulation of these opposing motors, which are often bound simultaneously to the same cargo. Recent progress demonstrates that organelle-associated scaffolding proteins, including Milton/TRAKs (trafficking kinesin-binding protein), JIP1, JIP3 (JNK-interacting proteins), huntingtin, and Hook1, interact with molecular motors to coordinate activity and sustain unidirectional transport. Scaffolding proteins also bind to upstream regulatory proteins, including kinases and GTPases, to modulate transport in the cell. This integration of regulatory control with motor activity allows for cargo-specific changes in the transport or targeting of organelles in response to cues from the complex cellular environment.

  1. (Online & Print): 2305-7432. htt

    African Journals Online (AJOL)

    Peter Berglez

    Factors affecting the growth of the Malawian film industry. Mufunanji Magalasi .... impact on Ghanaians reports on the role played by the British colonialists not only in .... Mpakati in the lobby of the Sheraton Hotel in Harare. Then the ..... Malawian vulnerable girl child, in the hands of the middle class, places the inspiration.

  2. Expansion of CAG triplet repeats by human DNA polymerases λ and β in vitro, is regulated by flap endonuclease 1 and DNA ligase 1.

    Science.gov (United States)

    Crespan, Emmanuele; Hübscher, Ulrich; Maga, Giovanni

    2015-05-01

    Huntington's disease (HD) is a neurological genetic disorder caused by the expansion of the CAG trinucleotide repeats (TNR) in the N-terminal region of coding sequence of the Huntingtin's (HTT) gene. This results in the addition of a poly-glutamine tract within the Huntingtin protein, resulting in its pathological form. The mechanism by which TRN expansion takes place is not yet fully understood. We have recently shown that DNA polymerase (Pol) β can promote the microhomology-mediated end joining and triplet expansion of a substrate mimicking a double strand break in the TNR region of the HTT gene. Here we show that TNR expansion is dependent on the structure of the DNA substrate, as well as on the two essential Pol β co-factors: flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). We found that Fen1 significantly stimulated TNR expansion by Pol β, but not by the related enzyme Pol λ, and subsequent ligation of the DNA products by Lig1. Interestingly, the deletion of N-terminal domains of Pol λ, resulted in an enzyme which displayed properties more similar to Pol β, suggesting a possible evolutionary mechanism. These results may suggest a novel mechanism for somatic TNR expansion in HD.

  3. Targeting several CAG expansion diseases by a single antisense oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Melvin M Evers

    Full Text Available To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUGn triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG(7, also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.

  4. Glyceraldehyde 3-phosphate dehydrogenase augments the intercellular transmission and toxicity of polyglutamine aggregates in a cell model of Huntington disease.

    Science.gov (United States)

    Mikhaylova, Elena R; Lazarev, Vladimir F; Nikotina, Alina D; Margulis, Boris A; Guzhova, Irina V

    2016-03-01

    The common feature of Huntington disease is the accumulation of oligomers or aggregates of mutant huntingtin protein (mHTT), which causes the death of a subset of striatal neuronal populations. The cytotoxic species can leave neurons and migrate to other groups of cells penetrating and damaging them in a prion-like manner. We hypothesized that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), previously shown to elevate the aggregation of mHTT, is associated with an increased efficiency of intercellular propagation of mHTT. GAPDH, on its own or together with polyglutamine species, was shown to be released into the extracellular milieu mainly from dying cells as assessed by a novel enzyme immunoassay, western blotting, and ultrafiltration. The conditioned medium of cells with growing GAPDH-polyQ aggregates was toxic to naïve cells, whereas depletion of the aggregates from the medium lowered this cytotoxicity. The GAPDH component of the aggregates was found to increase their toxicity by two-fold in comparison with polyQ alone. Furthermore, GAPDH-polyQ complexes were shown to penetrate acceptor cells and to increase the capacity of polyQ to prionize its intracellular homolog containing a repeat of 25 glutamine residues. Finally, inhibitors of intracellular transport showed that polyQ-GAPDH complexes, as well as GAPDH itself, penetrated cells using clathrin-mediated endocytosis. This suggested a pivotal role of the enzyme in the intercellular transmission of Huntington disease pathogenicity. In conclusion, GAPDH occurring in complexes with polyglutamine strengthens the prion-like activity and toxicity of the migrating aggregates. Aggregating polygluatmine tracts were shown to release from the cells over-expressing mutant huntingtin in a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The enzyme enhances the intracellular transport of aggregates to healthy cells, prionization of normal cellular proteins and finally cell death, thus

  5. Progress in studies of gene therapy for Huntington's disease

    Directory of Open Access Journals (Sweden)

    JIN Fan-ying

    2012-06-01

    Full Text Available Huntington's disease (HD is a kind of inherited neurodegenerative disorder characterized by movement problems, cognitive decline and psychiatry disturbance. HD is caused by mutation in gene IT -15 involving the expansion of a trinucleotide (CAG repeat encoding glutamine, which leads to abnormal conformation of huntingtin (Htt protein and finally emerge cytotoxic functions. Currently, HD remains a fatal untreatable disease. Gene therapy for HD discussed in this review is under preclinical studies. Silencing of mutant IT-15 via RNA interference (RNAi or antisense oligonucleotide (ASO has shown some effectiveness in mouse model studies. Increasing the clearance of mutant Htt protein could be achieved by viral-mediated delivery of anti-Htt intrabodies (iAbs or induction of autophagy, and beneficial results have been observed. Ectopic expression of neurotrophic factors, such as nerve growth factor (NGF and brain-derived neurotrophic factor (BDNF, mediated either by viral vectors or transplantation of genetically modified cells, has also been proved to be effective. Other gene-modifying methods aiming at correction of transcriptional dysregulation by histone modification, activation of endogenous neural stem cells, and normalization of calcium signaling and mitochondrial function, are also under intensive research. Gene therapy for Huntington's disease is promising, yet a long way remains from preclinical studies to clinical trials.

  6. Use of human stem cells in Huntington disease modeling and translational research.

    Science.gov (United States)

    Golas, Monika M; Sander, Bjoern

    2016-04-01

    Huntington disease (HD) is a devastating neurological disorder caused by an extended CAG repeat in exon 1 of the gene that encodes the huntingtin (HTT) protein. HD pathology involves a loss of striatal medium spiny neurons (MSNs) and progressive neurodegeneration affects the striatum and other brain regions. Because HTT is involved in multiple cellular processes, the molecular mechanisms of HD pathogenesis should be investigated on multiple levels. On the cellular level, in vitro stem cell models, such as induced pluripotent stem cells (iPSCs) derived from HD patients and HD embryonic stem cells (ESCs), have yielded progress. Approaches to differentiate functional MSNs from ESCs, iPSCs, and neural stem/progenitor cells (NSCs/NPCs) have been established, enabling MSN differentiation to be studied and disease phenotypes to be recapitulated. Isolation of target stem cells and precursor cells may also provide a resource for grafting. In animal models, transplantation of striatal precursors differentiated in vitro to the striatum has been reported to improve disease phenotype. Initial clinical trials examining intrastriatal transplantation of fetal neural tissue suggest a more favorable clinical course in a subset of HD patients, though shortcomings persist. Here, we review recent advances in the development of cellular HD models and approaches aimed at cell regeneration with human stem cells. We also describe how genome editing tools could be used to correct the HTT mutation in patient-specific stem cells. Finally, we discuss the potential and the remaining challenges of stem cell-based approaches in HD research and therapy development.

  7. Improvement of oxidative/anti-oxidative stress balance and expressions of NET and 5-HTT involved in the anti-depressive effect of reboxetine%改善氧化/抗氧化应激平衡及NET和5-HTT表达与瑞波西汀抗抑郁作用有关

    Institute of Scientific and Technical Information of China (English)

    李娜; 王涵; 文威; 周岐新

    2011-01-01

    Aim To investigate the relationship between the anti-depressive effect of reboxetine and the oxidative/anti-oxidative stress balance as well as expressions of NET and 5-HTT in rat depressive model induced by chronic unpredictable mild stress( CUMS ).Methods Sixty male SD rats were randomly divided into normal group( NG ). model group( MG ). reboxetine( 0.7 mg · kg-1 · d-1 )-treated normal group ( RNG), and reboxetine( 0. 7 mg·kg-1 · d-1 )-treated model group( RMG ). CUMS with solitary condition was taken to establish rat depression model. The openfield test and sucrose consumption were used to evaluate the depression behaviors of rats. Biochemistry methods were used to determine the content of MDA and activities of SOD and CAT in serum. Reverse transcription polymerase chain reaction( RT-PCR ) method was used to determine the mRNA expressions of NET and 5-HTT in either pons cerebelli or hippocampus tissues of rats. Hippocampal pathological change with HE staining slices was observed under microscope. Results It was shown that except MDA level,there were significant decreases of open-field scores( crossing, rearing, and preening activities ). sucrose consumption,SOD and CAT activities , and expressions of NET and 5-HTT, as well as obvious karyopyknosis and deep staining of hippocampal neurocytes in MG,in comparison with NG. The treatment of roboxetine obviously blunted the changes above caused by CUMS. However treatment of roboxetine had no influences on NG rats. Conclusions The treatment of reboxetine can improve depression behaviors of rats through reversing imbalance of oxidative/anti-oxidative stress systems and increasing expressions of NET and 5-HTT.%目的 探讨瑞波西汀抗慢性轻度不可预见性刺激(CUMS)致大鼠抑郁症行为与机体氧化/抗氧化应激平衡以及去甲肾上腺素转运体(NET)和5-羟色胺转运体(5-HTT)表达的关系.方法 ♂ SD大鼠60只,随机分为正常对照组(NG)、模型组(MG)、瑞波西汀(0.7 mg

  8. A novel mitochondrial tRNAAla gene variant causes chronic progressive external ophthalmoplegia in a patient with Huntington disease

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    Massimiliano Filosto

    2016-03-01

    Mitochondrial involvement is an emerging key determinant in the pathogenesis of Huntington disease and it is well known that mutant huntingtin influences the mitochondrial respiratory complexes II and III. A synergist effect of the HTT and MTTA mutations on respiratory chain function may be hypothesized in our patient and should be regarded as a spur for further studies on the mtDNA/HTT reciprocal interactions.

  9. Regulation of mRNA translation by MID1: a common mechanism of expanded CAG repeat RNAs

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    Nadine Griesche

    2016-10-01

    Full Text Available Expansion of CAG repeats, which code for the disease-causing polyglutamine protein, is a common feature in polyglutamine diseases. RNA-mediated mechanisms that contribute to neuropathology in polyglutamine diseases are important. RNA-toxicity describes a phenomenon by which the mutant CAG repeat RNA recruits RNA-binding proteins, thereby leading to aberrant function. For example the MID1 protein binds to mutant huntingtin (HTT RNA, which is linked to Huntington’s disease (HD, at its CAG repeat region and induces protein synthesis of mutant protein. But is this mechanism specific to HD or is it a common mechanism in CAG repeat expansion disorders? To answer this question, we have analysed the interaction between MID1 and three other CAG repeat mRNAs, Ataxin2 (ATXN2, Ataxin3 (ATXN3, and Ataxin7 (ATXN7, that all differ in the sequence flanking the CAG repeat. We show that ATXN2, ATXN3 and ATXN7 bind to MID1 in a CAG repeat length-dependent manner. Furthermore, we show that functionally, in line with what we have previously observed for HTT, the binding of MID1 to ATXN2, ATXN3 and ATXN7 mRNA induces protein synthesis in a repeat length-dependent manner. Our data suggest that regulation of protein translation by the MID1 complex is a common mechanism for CAG repeat containing mRNAs.

  10. Transgenic animal models for study of the pathogenesis of Huntington’s disease and therapy

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    Chang RB

    2015-04-01

    Full Text Available Renbao Chang,1 Xudong Liu,1 Shihua Li,2 Xiao-Jiang Li1,2 1State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People’s Republic of China; 2Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Abstract: Huntington’s disease (HD is caused by a genetic mutation that results in polyglutamine expansion in the N-terminal regions of huntingtin. As a result, this polyQ expansion leads to the misfolding and aggregation of mutant huntingtin as well as age-dependent neurodegeneration. The genetic mutation in HD allows for generating a variety of animal models that express different forms of mutant huntingtin and show differential pathology. Studies of these animal models have provided an important insight into the pathogenesis of HD. Mouse models of HD include transgenic mice, which express N-terminal or full-length mutant huntingtin ubiquitously or selectively in different cell types, and knock-in mice that express full-length mutant Htt at the endogenous level. Large animals, such as pig, sheep, and monkeys, have also been used to generate animal HD models. This review focuses on the different features of commonly used transgenic HD mouse models as well as transgenic large animal models of HD, and also discusses how to use them to identify potential therapeutics. Since HD shares many pathological features with other neurodegenerative diseases, identification of therapies for HD would also help to develop effective treatment for different neurodegenerative diseases that are also caused by protein misfolding and occur in an age-dependent manner. Keywords: transgenic animal models, Huntington’s disease, pathogenesis, therapy

  11. Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.

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    Bersuker, Kirill; Brandeis, Michael; Kopito, Ron R

    2016-04-25

    Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.

  12. Gene expression profiling of R6/2 transgenic mice with different CAG repeat lengths reveals genes associated with disease onset and progression in Huntington's disease.

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    Tang, Bin; Seredenina, Tamara; Coppola, Giovanni; Kuhn, Alexandre; Geschwind, Daniel H; Luthi-Carter, Ruth; Thomas, Elizabeth A

    2011-06-01

    R6/2 transgenic mice with expanded CAG repeats (>300) have a surprisingly prolonged disease progression and longer lifespan than prototypical parent R6/2 mice (carrying 150 CAGs); however, the mechanism of this phenotype amelioration is unknown. We compared gene expression profiles in the striatum of R6/2 transgenic mice carrying ~300 CAG repeats (R6/2(Q300) transgenic mice) to those carrying ~150 CAG repeats (R6/2(Q150) transgenic mice) and littermate wildtype controls in order to identify genes that may play determinant roles in the time course of phenotypic expression in these mice. Of the top genes showing concordant expression changes in the striatum of both R6/2 lines, 85% were decreased in expression, while discordant expression changes were observed mostly for genes upregulated in R6/2(Q300) transgenic mice. Upregulated genes in the R6/2(Q300) mice were associated with the ubiquitin ligase complex, cell adhesion, protein folding, and establishment of protein localization. We qPCR-validated increases in expression of genes related to the latter category, including Lrsam1, Erp29, Nasp, Tap1, Rab9b, and Pfdn5 in R6/2(Q300) mice, changes that were not observed in R6/2 mice with shorter CAG repeats, even in late stages (i.e., 12 weeks of age). We further tested Lrsam1 and Erp29, the two genes showing the greatest upregulation in R6/2(Q300) transgenic mice, for potential neuroprotective effects in primary striatal cultures overexpressing a mutated human huntingtin (htt) fragment. Overexpression of Lrsam1 prevented the loss of NeuN-positive cell bodies in htt171-82Q cultures, concomitant with a reduction of nuclear htt aggregates. Erp29 showed no significant effects in this model. This is consistent with the distinct pattern of htt inclusion localization observed in R6/2(Q300) transgenic mice, in which smaller cytoplasmic inclusions represent the major form of insoluble htt in the cell, as opposed to large nuclear inclusions observed in R6/2(Q150) transgenic mice

  13. Gene expression and behaviour in mouse models of HD.

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    Bowles, K R; Brooks, S P; Dunnett, S B; Jones, L

    2012-06-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disease, resulting in expansion of the CAG repeat in exon 1 of the HTT gene. The resulting mutant huntingtin protein has been implicated in the disruption of a variety of cellular functions, including transcription. Mouse models of HD have been central to the development of our understanding of gene expression changes in this disease, and are now beginning to elucidate the relationship between gene expression and behaviour. Here, we review current mouse models of HD and their characterisation in terms of gene expression. In addition, we look at how this can inform behaviours observed in mouse models of disease. The relationship between gene expression and behaviour in mouse models of HD is important, as this will further our knowledge of disease progression and its underlying molecular events, highlight new treatment targets, and potentially provide new biomarkers for therapeutic trials. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Neuroimmunology of Huntington’s Disease: Revisiting Evidence from Human Studies

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    Natalia P. Rocha

    2016-01-01

    Full Text Available Huntington’s disease (HD is a neurodegenerative disorder characterized by selective loss of neurons in the striatum and cortex, which leads to progressive motor dysfunction, cognitive decline, and psychiatric disorders. Although the cause of HD is well described—HD is a genetic disorder caused by a trinucleotide (CAG repeat expansion in the gene encoding for huntingtin (HTT on chromosome 4p16.3—the ultimate cause of neuronal death is still uncertain. Apart from impairment in systems for handling abnormal proteins, other metabolic pathways and mechanisms might contribute to neurodegeneration and progression of HD. Among these, inflammation seems to play a role in HD pathogenesis. The current review summarizes the available evidence about immune and/or inflammatory changes in HD. HD is associated with increased inflammatory mediators in both the central nervous system and periphery. Accordingly, there have been some attempts to slow HD progression targeting the immune system.

  15. Human glia can both induce and rescue aspects of disease phenotype in Huntington disease

    DEFF Research Database (Denmark)

    Benraiss, Abdellatif; Wang, Su; Herrlinger, Stephanie

    2016-01-01

    (hGPCs), derived from either human embryonic stem cells or mHTT-transduced fetal hGPCs. Here we show that mHTT glia can impart disease phenotype to normal mice, since mice engrafted intrastriatally with mHTT hGPCs exhibit worse motor performance than controls, and striatal neurons in mHTT glial......The causal contribution of glial pathology to Huntington disease (HD) has not been heavily explored. To define the contribution of glia to HD, we established human HD glial chimeras by neonatally engrafting immunodeficient mice with mutant huntingtin (mHTT)-expressing human glial progenitor cells...... survival in R6/2 HD mice. These observations suggest a causal role for glia in HD, and further suggest a cell-based strategy for disease amelioration in this disorder....

  16. Huntington disease in the South African population occurs on diverse and ethnically distinct genetic haplotypes

    NARCIS (Netherlands)

    Baine, Fiona K.; Kay, Chris; Ketelaar, Maria E.; Collins, Jennifer A.; Semaka, Alicia; Doty, Crystal N.; Krause, Amanda; Greenberg, L. Jacquie; Hayden, Michael R.

    2013-01-01

    Huntington disease (HD) is a neurodegenerative disorder resulting from the expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene. Worldwide prevalence varies geographically with the highest figures reported in populations of European ancestry. HD in South Africa has been reported in C

  17. Reversal Learning and Associative Memory Impairments in a BACHD Rat Model for Huntington Disease

    NARCIS (Netherlands)

    Abada, Yah-se K.; Nguyen, Huu Phuc; Ellenbroek, Bart; Schreiber, Rudy

    2013-01-01

    Chorea and psychiatric symptoms are hallmarks of Huntington disease (HD), a neurodegenerative disorder, genetically characterized by the presence of expanded CAG repeats (>35) in the HUNTINGTIN (HTT) gene. HD patients present psychiatric symptoms prior to the onset of motor symptoms and we recently

  18. Ubiquitin-proteasome system involvement in Huntington’s disease

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    Zaira eOrtega

    2014-09-01

    Full Text Available Huntington’s disease (HD is a genetic autosomal dominant neurodegenerative disease caused by the expansion of a CAG repeat in the huntingtin (htt gene. This triplet expansion encodes a polyglutamine stretch (polyQ in the N-terminus of the high molecular weight (348-kDa and ubiquitously expressed protein huntingtin (htt. Normal individuals have between 6 and 35 CAG triplets, while expansions longer than 40 repeats lead to HD. The onset and severity of the disease depend on the length of the polyQ tract: the longer the polyQ is, the earlier the disease begins and the more severe the symptoms are. One of the main histopathological hallmarks of HD is the presence of intraneuronal proteinaceous inclusion bodies (IBs, whose prominent and invariant feature is the presence of Ubiquitin (Ub; therefore, they can be detected with anti-ubiquitin and anti-proteasome antibodies. This, together with the observation that mutations in components of the Ubiquitin Proteasome system (UPS give rise to some neurodegenerative diseases, suggests that UPS impairment may be causative of HD. Even though the link between disrupted Ub homeostasis and protein aggregation to HD is undisputed, the functional significance of these correlations and their mechanistic implications remains unresolved. Moreover, there is no consistent evidence documenting an accompanying decrease in levels of free Ub or disruption of Ub pool dynamics in neurodegenerative disease or models thus suggesting that the Ub-conjugate accumulation may be benign and just underlie lesion in 26S function. In this chapter we will elaborate on the different studies that have been performed using different experimental approaches, in order to shed light to this matter.

  19. CAMELOT: A machine learning approach for coarse-grained simulations of aggregation of block-copolymeric protein sequences

    Energy Technology Data Exchange (ETDEWEB)

    Ruff, Kiersten M. [Computational and Systems Biology Program and Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, Missouri 63130-4899 (United States); Harmon, Tyler S. [Department of Physics and Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, Missouri 63130-4899 (United States); Pappu, Rohit V., E-mail: pappu@wustl.edu [Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University in St. Louis, CB 1097, St. Louis, Missouri 63130-4899 (United States)

    2015-12-28

    We report the development and deployment of a coarse-graining method that is well suited for computer simulations of aggregation and phase separation of protein sequences with block-copolymeric architectures. Our algorithm, named CAMELOT for Coarse-grained simulations Aided by MachinE Learning Optimization and Training, leverages information from converged all atom simulations that is used to determine a suitable resolution and parameterize the coarse-grained model. To parameterize a system-specific coarse-grained model, we use a combination of Boltzmann inversion, non-linear regression, and a Gaussian process Bayesian optimization approach. The accuracy of the coarse-grained model is demonstrated through direct comparisons to results from all atom simulations. We demonstrate the utility of our coarse-graining approach using the block-copolymeric sequence from the exon 1 encoded sequence of the huntingtin protein. This sequence comprises of 17 residues from the N-terminal end of huntingtin (N17) followed by a polyglutamine (polyQ) tract. Simulations based on the CAMELOT approach are used to show that the adsorption and unfolding of the wild type N17 and its sequence variants on the surface of polyQ tracts engender a patchy colloid like architecture that promotes the formation of linear aggregates. These results provide a plausible explanation for experimental observations, which show that N17 accelerates the formation of linear aggregates in block-copolymeric N17-polyQ sequences. The CAMELOT approach is versatile and is generalizable for simulating the aggregation and phase behavior of a range of block-copolymeric protein sequences.

  20. The Copper Metabolism MURR1 domain protein 1 (COMMD1 modulates the aggregation of misfolded protein species in a client-specific manner.

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    Willianne I M Vonk

    Full Text Available The Copper Metabolism MURR1 domain protein 1 (COMMD1 is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α. Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1 and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS, cause misfolding and aggregation of the mutant SOD1 (mSOD1 protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

  1. The Copper Metabolism MURR1 domain protein 1 (COMMD1) modulates the aggregation of misfolded protein species in a client-specific manner.

    Science.gov (United States)

    Vonk, Willianne I M; Kakkar, Vaishali; Bartuzi, Paulina; Jaarsma, Dick; Berger, Ruud; Hofker, Marten H; Klomp, Leo W J; Wijmenga, Cisca; Kampinga, Harm H; van de Sluis, Bart

    2014-01-01

    The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

  2. Chapter 3: A fluorescent window into protein folding and aggregation in cells.

    Science.gov (United States)

    Ignatova, Zoya; Gierasch, Lila M

    2008-01-01

    Evolutionary selective pressures have tuned the efficiency of the protein-folding reaction in the crowded complex environment in the cell. Nevertheless, the fidelity of folding is imperfect, leading to off-pathway intermolecular interactions that compete with proper folding and to consequent formation of thermodynamically stable aggregates. Such aggregates constitute the histopathological hallmarks of many neurodegenerative pathologies. Yet, most of the approaches to characterize protein folding and/or misfolding are limited to in vitro conditions. Here, we describe a strategy to directly monitor the behavior of a protein in prokaryotic and eukaryotic cells. The method is based on incorporation of structurally non-perturbing, specific binding motifs for a bis-arsenical fluoroscein dye, FlAsH, in sites that result in distinct dye fluorescence signals for the folded and unfolded states of the protein under study. Our approach has been developed using as a case study the predominantly beta-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein, alone or as a chimera fused to the exon 1-encoded fragment of huntingtin, which harbors a polyglutamine repeat tract. We have designed protocols to label this protein in vivo and to monitor the resulting fluorescence signal, which reports on any misfolding transition and formation of aggregates, yielding quantitatively interpretable data.

  3. Pathogenic protein seeding in Alzheimer disease and other neurodegenerative disorders.

    Science.gov (United States)

    Jucker, Mathias; Walker, Lary C

    2011-10-01

    The misfolding and aggregation of specific proteins is a seminal occurrence in a remarkable variety of neurodegenerative disorders. In Alzheimer disease (the most prevalent cerebral proteopathy), the two principal aggregating proteins are β-amyloid (Aβ) and tau. The abnormal assemblies formed by conformational variants of these proteins range in size from small oligomers to the characteristic lesions that are visible by optical microscopy, such as senile plaques and neurofibrillary tangles. Pathologic similarities with prion disease suggest that the formation and spread of these proteinaceous lesions might involve a common molecular mechanism-corruptive protein templating. Experimentally, cerebral β-amyloidosis can be exogenously induced by exposure to dilute brain extracts containing aggregated Aβ seeds. The amyloid-inducing agent probably is Aβ itself, in a conformation generated most effectively in the living brain. Once initiated, Aβ lesions proliferate within and among brain regions. The induction process is governed by the structural and biochemical nature of the Aβ seed, as well as the attributes of the host, reminiscent of pathogenically variant prion strains. The concept of prionlike induction and spreading of pathogenic proteins recently has been expanded to include aggregates of tau, α-synuclein, huntingtin, superoxide dismutase-1, and TDP-43, which characterize such human neurodegenerative disorders as frontotemporal lobar degeneration, Parkinson/Lewy body disease, Huntington disease, and amyotrophic lateral sclerosis. Our recent finding that the most effective Aβ seeds are small and soluble intensifies the search in bodily fluids for misfolded protein seeds that are upstream in the proteopathic cascade, and thus could serve as predictive diagnostics and the targets of early, mechanism-based interventions. Establishing the clinical implications of corruptive protein templating will require further mechanistic and epidemiologic investigations

  4. Expression of expanded CAG transcripts triggers nucleolar stress in Huntington's disease.

    Science.gov (United States)

    Tsoi, Ho; Chan, Ho Yin Edwin

    2013-06-01

    Polyglutamine (polyQ) diseases, including several types of spinocerebellar ataxias and Huntington's disease (HD), are dominantly inherited neurodegenerative disorders caused by the expansion of the glutamine-coding CAG repeat in the open reading frame of the disease gene. Apart from being translated to produce toxic elongated polyQ domain-containing disease proteins, transcribed expanded CAG RNAs per se also exert toxicity in polyQ degeneration. In the R6/2 HD transgenic mouse model, expanded mutant Huntingtin (Htt) transcripts were found to physically interact with nucleolin (NCL), a nucleolar protein that plays a crucial role in ribosome biogenesis. We further demonstrated that mutant Htt transcripts deprived NCL from binding onto the Upstream Control Element (UCE) of the ribosomal RNA (rRNA) promoter. This resulted in UCE hypermethylation which abolished the binding of the transcription factor Upstream Binding Factor to UCE and subsequently led to down-regulation of pre-45s rRNA transcription. We also found that the p53/mitochondria-dependent nucleolar stress cell death pathway was activated in polyQ diseases. Ribosomal RNA transcription dysfunction has been reported in other types of neurodegenerative disorders including Alzheimer's disease; it is anticipated that nucleolar stress is one common pathogenic signaling mechanism shared by different forms of neurodegeneration.

  5. Atrophin-1, the DRPLA gene product, interacts with two families of WW domain-containing proteins.

    Science.gov (United States)

    Wood, J D; Yuan, J; Margolis, R L; Colomer, V; Duan, K; Kushi, J; Kaminsky, Z; Kleiderlein, J J; Sharp, A H; Ross, C A

    1998-06-01

    Atrophin-1 contains a polyglutamine repeat, expansion of which is responsible for dentatorubral and pallidoluysian atrophy (DRPLA). The normal function of atrophin-1 is unknown. We have identified five atrophin-1 interacting proteins (AIPs) which bind to atrophin-1 in the vicinity of the polyglutamine tract using the yeast two-hybrid system. Four of the interactions were confirmed using in vitro binding assays. All five interactors contained multiple WW domains. Two are novel. The AIPs can be divided into two distinct classes. AIP1 and AIP3/WWP3 are MAGUK-like multidomain proteins containing a number of protein-protein interaction modules, namely a guanylate kinase-like region, two WW domains, and multiple PDZ domains. AIP2/WWP2, AIP4, and AIP5/WWP1 are highly homologous, each having four WW domains and a HECT domain characteristic of ubiquitin ligases. These interactors are similar to recently isolated huntingtin-interacting proteins, suggesting possible commonality of function between two proteins responsible for very similar diseases.

  6. ANIMAL MODELS FOR HUNTINGTON’S DISEASES: A REVIEW

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    Sharma Manisha

    2012-10-01

    Full Text Available Huntington's disease (HD is an inherited autosomal, progressive neurodegenerative disorder associated with involuntary abnormal movements (chorea, cognitive impairments and psychiatric disturbances. HD is caused by an abnormal expansion of a CAG region located in exon 1 of the gene encoding the huntingtin protein (Htt and is the causative factor in the pathogenesis of HD Animal models of HD have provided insight into disease pathology and the outcomes of thera- peutic strategies. Earlier studies of HD most often used toxin-induced models to study mitochondrial impairment and excitotoxicity-induced cell death, which are both mechanisms of degeneration seen in the HD brain. These models, based on 3-nitropropionic acid and quinolinic acid, respectively, are still often used in HD studies. The discovery in 1993 of the huntingtin mutation led to the creation of newer models that incorporate a similar genetic defect. These models, which include transgenic and knock-in rodents, are more representative of the HD progression and pathology. An even more recent model that uses a ovine transgenic model (sheep model,fly models ,cell cultures models for better understanding of gene mutation in and in mammalian and nonhuman primates, as it is difficult to produce genetic models in these species. This article examines the aforementioned models and describes their use in HD research, including aspects of the creation, de- livery, pathology, and tested therapies for each model.

  7. Microglial Activation in the Pathogenesis of Huntington's Disease.

    Science.gov (United States)

    Yang, Hui-Ming; Yang, Su; Huang, Shan-Shan; Tang, Bei-Sha; Guo, Ji-Feng

    2017-01-01

    Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by expanded CAG trinucleotide repeats (>36) in exon 1 of HTT gene that encodes huntingtin protein. Although HD is characterized by a predominant loss of neurons in the striatum and cortex, previous studies point to a critical role of aberrant accumulation of mutant huntingtin in microglia that contributes to the progressive neurodegeneration in HD, through both cell-autonomous and non-cell-autonomous mechanisms. Microglia are resident immune cells in the central nervous system (CNS), which function to surveil the microenvironment at a quiescent state. In response to various pro-inflammatory stimuli, microglia become activated and undergo two separate phases (M1 and M2 phenotype), which release pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), anti-inflammatory cytokines, and growth factors (TGF-β, CD206, and Arg1), respectively. Immunoregulation by microglial activation could be either neurotoxic or neuroprotective. In this review, we summarized current understanding about microglial activation in the pathogenesis and progression of HD, with a primary focus of M1 and M2 phenotype of activated microglia and their corresponding signaling pathways.

  8. Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant.

    Science.gov (United States)

    Ignatova, Zoya; Gierasch, Lila M

    2006-09-05

    Small organic molecules termed osmolytes are harnessed by a variety of cell types in a wide range of organisms to counter unfavorable physiological conditions that challenge protein stability and function. Using a well characterized reporter system that we developed to allow in vivo observations, we have explored how the osmolyte proline influences the stability and aggregation of a model aggregation-prone protein, P39A cellular retinoic acid-binding protein. Strikingly, we find that the natural osmolyte proline abrogates aggregation both in vitro and in vivo (in an Escherichia coli expression system). Importantly, proline also prevented aggregation of constructs containing exon 1 of huntingtin with extended polyglutamine tracts. Although compatible osmolytes are known to stabilize the native state, our results point to a destabilizing effect of proline on partially folded states and early aggregates and a solubilizing effect on the native state. Because proline is believed to act through a combination of solvophobic backbone interactions and favorable side-chain interactions that are not specific to a particular sequence or structure, the observed effect is likely to be general. Thus, the osmolyte proline may be protective against biomedically important protein aggregates that are hallmarks of several late-onset neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. In addition, these results should be of practical importance because they may enable protein expression at higher efficiency under conditions where aggregation competes with proper folding.

  9. The iron regulatory capability of the major protein participants in prevalent neurodegenerative disorders

    Directory of Open Access Journals (Sweden)

    Bruce Xue Wen Wong

    2014-04-01

    Full Text Available As with most bioavailable transition metals, iron is essential for many metabolic processes required by the cell but when left unregulated is implicated as a potent source of reactive oxygen species. It is uncertain whether the brain’s evident vulnerability to reactive species-induced oxidative stress is caused by a reduced capability in cellular response or an increased metabolic activity. Either way, dys-regulated iron levels appear to be involved in oxidative stress provoked neurodegeneration. As in peripheral iron management, cells within the central nervous system tightly regulate iron homeostasis via responsive expression of select proteins required for iron flux, transport and storage. Recently proteins directly implicated in the most prevalent neurodegenerative diseases, such as amyloid-β precursor protein, tau, α-synuclein, prion protein and huntingtin, have been connected to neuronal iron homeostatic control. This suggests that disrupted expression, processing or location of these proteins may result in a failure of their cellular iron homeostatic roles and augment the common underlying susceptibility to neuronal oxidative damage that is triggered in neurodegenerative disease.

  10. Neural and mesenchymal stem cells in animal models of Huntington's disease: past experiences and future challenges.

    Science.gov (United States)

    Kerkis, Irina; Haddad, Monica Santoro; Valverde, Cristiane Wenceslau; Glosman, Sabina

    2015-12-14

    Huntington's disease (HD) is an inherited disease that causes progressive nerve cell degeneration. It is triggered by a mutation in the HTT gene that strongly influences functional abilities and usually results in movement, cognitive and psychiatric disorders. HD is incurable, although treatments are available to help manage symptoms and to delay the physical, mental and behavioral declines associated with the condition. Stem cells are the essential building blocks of life, and play a crucial role in the genesis and development of all higher organisms. Ablative surgical procedures and fetal tissue cell transplantation, which are still experimental, demonstrate low rates of recovery in HD patients. Due to neuronal cell death caused by accumulation of the mutated huntingtin (mHTT) protein, it is unlikely that such brain damage can be treated solely by drug-based therapies. Stem cell-based therapies are important in order to reconstruct damaged brain areas in HD patients. These therapies have a dual role: stem cell paracrine action, stimulating local cell survival, and brain tissue regeneration through the production of new neurons from the intrinsic and likely from donor stem cells. This review summarizes current knowledge on neural stem/progenitor cell and mesenchymal stem cell transplantation, which has been carried out in several animal models of HD, discussing cell distribution, survival and differentiation after transplantation, as well as functional recovery and anatomic improvements associated with these approaches. We also discuss the usefulness of this information for future preclinical and clinical studies in HD.

  11. The Role of the Immune System in Huntington’s Disease

    Directory of Open Access Journals (Sweden)

    Gisa Ellrichmann

    2013-01-01

    Full Text Available Huntington’s disease (HD is characterized by a progressive course of disease until death 15–20 years after the first symptoms occur and is caused by a mutation with expanded CAG repeats in the huntingtin (htt protein. Mutant htt (mhtt in the striatum is assumed to be the main reason for neurodegeneration. Knowledge about pathophysiology has rapidly improved discussing influences of excitotoxicity, mitochondrial damage, free radicals, and inflammatory mechanisms. Both innate and adaptive immune systems may play an important role in HD. Activation of microglia with expression of proinflammatory cytokines, impaired migration of macrophages, and deposition of complement factors in the striatum indicate an activation of the innate immune system. As part of the adaptive immune system, dendritic cells (DCs prime T-cell responses secreting inflammatory mediators. In HD, DCs may contain mhtt which brings the adaptive immune system into the focus of interest. These data underline an increasing interest in the peripheral immune system for pathomechanisms of HD. It is still unclear if neuroinflammation is a reactive process or if there is an active influence on disease progression. Further understanding the influence of inflammation in HD using mouse models may open various avenues for promising therapeutic approaches aiming at slowing disease progression or forestalling onset of disease.

  12. Influence of Species Differences on the Neuropathology of Transgenic Huntington's Disease Animal Models

    Institute of Scientific and Technical Information of China (English)

    Xiao-Jiang Li; Shihua Li

    2012-01-01

    Transgenic animal models have revealed much about the pathogenesis of age-dependent neurodegenerative diseases and proved to be a useful tool for uncovering therapeutic targets.Huntington's disease is a well-characterized neurodegenerative disorder that is caused by expansion of a CAG repeat,which results in expansion of a polyglutamine tract in the N-terminal region of huntingtin (HTT).Similar CAG/glutamine expansions are also found to cause eight other neurodegenerative diseases that affect distinct brain regions in an agedependent manner.Identification of this CAG/glutamine expansion has led to the generation of a variety of transgenic animal models.Of these different animal models,transgenic mice have been investigated extensively,and they show similar neuropathology and phenotypes as seen in their respective diseases.The common pathological hallmark of age-dependent neurodegeneration is the formation of aggregates or inclusions consisting of misfolded proteins in the affected brain regions; however,overt or striking neurodegeneration and apoptosis have not been reported in most transgenic mouse models for age-dependent diseases,including HD.By comparing the neuropathology of transgenic HD mouse,pig,and monkey models,we found that mutant HTT is more toxic to larger animals than mice,and larger animals also show neuropathology that has not been uncovered by transgenic mouse models.This review will discuss the importancc of transgenic large animal models for analyzing the pathogenesis of neurodegenerative diseases and developing effective treatments.

  13. Loss aversion and 5HTT gene variants in adolescent anxiety

    Directory of Open Access Journals (Sweden)

    Monique Ernst

    2014-04-01

    Full Text Available Loss aversion, a well-documented behavioral phenomenon, characterizes decisions under risk in adult populations. As such, loss aversion may provide a reliable measure of risky behavior. Surprisingly, little is known about loss aversion in adolescents, a group who manifests risk-taking behavior, or in anxiety disorders, which are associated with risk-avoidance. Finally, loss aversion is expected to be modulated by genotype, particularly the serotonin transporter (SERT gene variant, based on its role in anxiety and impulsivity. This genetic modulation may also differ between anxious and healthy adolescents, given their distinct propensities for risk taking. The present work examines the modulation of loss aversion, an index of risk-taking, and reaction-time to decision, an index of impulsivity, by the serotonin-transporter-gene-linked polymorphisms (5HTTLPR in healthy and clinically anxious adolescents. Findings show that loss aversion (1 does manifest in adolescents, (2 does not differ between healthy and clinically anxious participants, and (3, when stratified by SERT genotype, identifies a subset of anxious adolescents who are high SERT-expressers, and show excessively low loss-aversion and high impulsivity. This last finding may serve as preliminary evidence for 5HTTLPR as a risk factor for the development of comorbid disorders associated with risk-taking and impulsivity in clinically anxious adolescents.

  14. RESEARCH OF GENETIC POLYMORPHISM OF 5-HTT IN CHILDHOOD AUTISM

    Institute of Scientific and Technical Information of China (English)

    Sun Xiaomian; Li Yamei; Zheng Chongxun

    2006-01-01

    Objective To reveal the relationship between the 5-HTTLPR and the Chinese Han nationality children with CA, compared the distribution of the 5-HTTLPR between the Han Chinese children with CA and healthy Han Chinese children ,and analyzed the association between the 5-HTTLPR and clinical symptoms of the Han Chinese children with CA. Methods Genomic DNAs of fifty subjects including 25 autistic children and 25 controls were extracted from blood samples. PCR amplification using Oligonucleotide primers flanking 5-HTTLPR was performed. Results ① Three kinds of alleles including the S (short) allele, the L (long) allele and the VL allele were found , and the 5-HTTLPR genotypes shown were S/S, L/L, S/L and L/VL. ② Allele frequencies did not differ significantly in patient groups in comparison with the control sample. No significant difference was identified between the observed 5-HTTLPR genotype distribution of the patient groups and control group. ③ The distribution of homozygous and heterozygous subjects between the two groups differed significantly. ④ The genotypes of the 5-HTTLPR polymorphism correlated significantly with the Body Movement Factor. ⑤ The allele frequency of healthy Han Chinese population and that of healthy Japanese population were similar. The frequency of S allele in not only autistic subjects but also healthy children in this study was considerably more than that in Caucasians and the frequency of L allele in our subjects decreased correspondingly. Conclusion ① A significant difference in the allele frequency between the Han Chinese and Caucasian populations was found. ② The genotypes of the 5-HTTLPR polymorphism correlated significantly with the Body Movement Factor of the patients. ③ The homozygote and the L allele were positively relevant to CA and they might be the risk factors of CA. The heterozygote and the S allele were negatively relevant to CA and they might be the protective factors of CA.

  15. RESEARCH OF GENETIC POLYMORPHISM OF 5-HTT IN CHILDHOOD AUTISM

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Childhood autis m(CA)is a kind of severeneurodevelopmental disorder usually occurringwithin the first3years of life,and it is character-ized by qualitative i mpair ments in reciprocal socialinteraction and communication,deviant languageand restricted and stereotyped patterns of interestsand activities.The latest epidemiological investiga-tion shows that the population prevalence of autis mreaches1/1000-1/2500[1].The ratio of affectedmales to females is4-5∶1in most studies whilethe ratio of6.5-9∶1is reported...

  16. Protein oxidation in Huntington disease affects energy production and vitamin B6 metabolism.

    Science.gov (United States)

    Sorolla, Ma Alba; Rodríguez-Colman, Ma José; Tamarit, Jordi; Ortega, Zaira; Lucas, José J; Ferrer, Isidre; Ros, Joaquim; Cabiscol, Elisa

    2010-08-15

    Huntington disease (HD) is an inherited neurodegenerative disorder that initially affects the striatum and progressively the cortex. Oxidative stress in HD has been described as important to disease progression. In this study, protein carbonylation, used as a marker of protein oxidation, was analyzed in human brain striatum. A comparison of HD samples to matched controls identified 13 carbonylated proteins, including enzymes involved in the glycolytic pathway and mitochondrial proteins related to ATP production. Oxidation of the mitochondrial enzymes resulted in decreased catalytic activity, in good agreement with the energy deficiency observed in HD. We also found carbonylation of pyridoxal kinase and antiquitin 1, both involved in the metabolism of pyridoxal 5-phosphate, the active form of vitamin B6. The Tet/HD94 conditional mouse model allowed us to demonstrate that increased carbonylation in striatum is dependent on mutant huntingtin expression. As in humans, pyridoxal kinase showed decreased levels and was highly carbonylated in the gene-on mice; these modifications were reverted in the gene-off mice. We hypothesize that both pyridoxal kinase and antiquitin 1 oxidation could result in decreased pyridoxal 5-phosphate availability necessary as a cofactor in transaminations, synthesis of glutathione, and synthesis of GABA and dopamine, two neurotransmitters that play a key role in HD pathology.

  17. Prefoldin Protects Neuronal Cells from Polyglutamine Toxicity by Preventing Aggregation Formation*

    Science.gov (United States)

    Tashiro, Erika; Zako, Tamotsu; Muto, Hideki; Itoo, Yoshinori; Sörgjerd, Karin; Terada, Naofumi; Abe, Akira; Miyazawa, Makoto; Kitamura, Akira; Kitaura, Hirotake; Kubota, Hiroshi; Maeda, Mizuo; Momoi, Takashi; Iguchi-Ariga, Sanae M. M.; Kinjo, Masataka; Ariga, Hiroyoshi

    2013-01-01

    Huntington disease is caused by cell death after the expansion of polyglutamine (polyQ) tracts longer than ∼40 repeats encoded by exon 1 of the huntingtin (HTT) gene. Prefoldin is a molecular chaperone composed of six subunits, PFD1–6, and prevents misfolding of newly synthesized nascent polypeptides. In this study, we found that knockdown of PFD2 and PFD5 disrupted prefoldin formation in HTT-expressing cells, resulting in accumulation of aggregates of a pathogenic form of HTT and in induction of cell death. Dead cells, however, did not contain inclusions of HTT, and analysis by a fluorescence correlation spectroscopy indicated that knockdown of PFD2 and PFD5 also increased the size of soluble oligomers of pathogenic HTT in cells. In vitro single molecule observation demonstrated that prefoldin suppressed HTT aggregation at the small oligomer (dimer to tetramer) stage. These results indicate that prefoldin inhibits elongation of large oligomers of pathogenic Htt, thereby inhibiting subsequent inclusion formation, and suggest that soluble oligomers of polyQ-expanded HTT are more toxic than are inclusion to cells. PMID:23720755

  18. From the test tube to the cell: exploring the folding and aggregation of a beta-clam protein.

    Science.gov (United States)

    Ignatova, Zoya; Krishnan, Beena; Bombardier, Jeffrey P; Marcelino, Anna Marie C; Hong, Jiang; Gierasch, Lila M

    2007-01-01

    A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model beta-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.

  19. Identification of oral cancer related candidate genes by integrating protein-protein interactions, gene ontology, pathway analysis and immunohistochemistry.

    Science.gov (United States)

    Kumar, Ravindra; Samal, Sabindra K; Routray, Samapika; Dash, Rupesh; Dixit, Anshuman

    2017-05-30

    In the recent years, bioinformatics methods have been reported with a high degree of success for candidate gene identification. In this milieu, we have used an integrated bioinformatics approach assimilating information from gene ontologies (GO), protein-protein interaction (PPI) and network analysis to predict candidate genes related to oral squamous cell carcinoma (OSCC). A total of 40973 PPIs were considered for 4704 cancer-related genes to construct human cancer gene network (HCGN). The importance of each node was measured in HCGN by ten different centrality measures. We have shown that the top ranking genes are related to a significantly higher number of diseases as compared to other genes in HCGN. A total of 39 candidate oral cancer target genes were predicted by combining top ranked genes and the genes corresponding to significantly enriched oral cancer related GO terms. Initial verification using literature and available experimental data indicated that 29 genes were related with OSCC. A detailed pathway analysis led us to propose a role for the selected candidate genes in the invasion and metastasis in OSCC. We further validated our predictions using immunohistochemistry (IHC) and found that the gene FLNA was upregulated while the genes ARRB1 and HTT were downregulated in the OSCC tissue samples.

  20. Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay.

    Science.gov (United States)

    Röthlein, Christoph; Miettinen, Markus S; Borwankar, Tejas; Bürger, Jörg; Mielke, Thorsten; Kumke, Michael U; Ignatova, Zoya

    2014-09-26

    The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered β-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure.

  1. Silencing Huntington's chorea: Is RNA Interference a Potential Cure?

    Directory of Open Access Journals (Sweden)

    Gerlinde A. Metz

    2006-01-01

    Full Text Available In 1872, George Huntington described Huntington's disease as characterized by motor, cognitive and psychiatric impairments. Huntington's disease is a dominant and autosomal mutation on chromosome 4 featuring the insertion of numerous CAG repeats. CAG codes for the amino acid, glutmanine that forms part of the Huntingtin protein (htt. Excess glutamine attachments make htt prone to accumulate in neurons. Three genes can be considered when developing therapies for Huntington's disease. They include targeting the symptoms of the disease, the progression of the disease and the cause of the disease. By using RNA interference (RNAi, the cause of the disease can be targeted. RNAi is a method that could potentially silence the formation of abnormal htt. This paper will discuss how RNAi could potentially cure Huntington's disease, by describing the genetic and proteinomic basis of Huntington's disease, the function of RNAi in Huntington's disease and the problems of benefits of RNAi. Preliminary work using RNAi in transgenic mice has shown a decrease in the behavioural expression of the mutant Huntington gene. There are several limitations associated with using RNAi as a gene therapy. For example, the effects of RNAi are short lived. A transposition system such as Sleeping Beauty can be used to increase the integration of the gene, however, for patients who currently have Huntington's disease, RNAi may potentially be used in combination with drugs or other treatments to target both symptoms and the underlying cause of Huntington's disease. This combination could eventually alleviate many painful symptoms associated with Huntington's disease and could even stop the progressive neurodegeneration of Huntington's disease. This review concludes that a substantial amount of new research is still necessary before RNAi is directly applicable to human patients with Huntington's disease.

  2. Striatal synaptic dysfunction and hippocampal plasticity deficits in the Hu97/18 mouse model of Huntington disease.

    Directory of Open Access Journals (Sweden)

    Karolina Kolodziejczyk

    Full Text Available Huntington disease (HD is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene (HTT encoding the huntingtin protein (HTT. This mutation leads to multiple cellular and synaptic alterations that are mimicked in many current HD animal models. However, the most commonly used, well-characterized HD models do not accurately reproduce the genetics of human disease. Recently, a new 'humanized' mouse model, termed Hu97/18, has been developed that genetically recapitulates human HD, including two human HTT alleles, no mouse Hdh alleles and heterozygosity of the HD mutation. Previously, behavioral and neuropathological testing in Hu97/18 mice revealed many features of HD, yet no electrophysiological measures were employed to investigate possible synaptic alterations. Here, we describe electrophysiological changes in the striatum and hippocampus of the Hu97/18 mice. At 9 months of age, a stage when cognitive deficits are fully developed and motor dysfunction is also evident, Hu97/18 striatal spiny projection neurons (SPNs exhibited small changes in membrane properties and lower amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs; however, release probability from presynaptic terminals was unaltered. Strikingly, these mice also exhibited a profound deficiency in long-term potentiation (LTP at CA3-to-CA1 synapses. In contrast, at 6 months of age we found only subtle alterations in SPN synaptic transmission, while 3-month old animals did not display any electrophysiologically detectable changes in the striatum and CA1 LTP was intact. Together, these data reveal robust, progressive deficits in synaptic function and plasticity in Hu97/18 mice, consistent with previously reported behavioral abnormalities, and suggest an optimal age (9 months for future electrophysiological assessment in preclinical studies of HD.

  3. Ca2+ Handling in Isolated Brain Mitochondria and Cultured Neurons Derived from the YAC128 Mouse Model of Huntington’s Disease

    Science.gov (United States)

    Pellman, Jessica J.; Hamilton, James; Brustovetsky, Tatiana; Brustovetsky, Nickolay

    2015-01-01

    We investigated Ca2+ handling in isolated brain synaptic and nonsynaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington’s disease (HD). Both synaptic and nonsynaptic mitochondria from 2- and 12-month-old YAC128 mice had larger Ca2+ uptake capacity than mitochondria from YAC18 and wild-type FVB/NJ mice. Synaptic mitochondria from 12-month-old YAC128 mice had further augmented Ca2+ capacity compared with mitochondria from 2-month-old YAC128 mice and age-matched YAC18 and FVB/NJ mice. This increase in Ca2+ uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12-month-old YAC128 mice. We speculate that this may happen due to mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca2+-induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100μM glutamate produced transient elevations in cytosolic Ca2+ followed by recovery to near resting levels. Following recovery of cytosolic Ca2+, mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca2+, suggesting similar Ca2+ release and, consequently, Ca2+ loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca2+ handling in brain mitochondria of YAC128 mice. PMID:25963273

  4. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... for the vegetarian proteins, whether they have carbohydrate. Protein Choices Plant-Based Proteins Plant-based protein foods ...

  5. Monomeric, Oligomeric and Polymeric Proteins in Huntington Disease and Other Diseases of Polyglutamine Expansion

    Directory of Open Access Journals (Sweden)

    Guylaine Hoffner

    2014-03-01

    Full Text Available Huntington disease and other diseases of polyglutamine expansion are each caused by a different protein bearing an excessively long polyglutamine sequence and are associated with neuronal death. Although these diseases affect largely different brain regions, they all share a number of characteristics, and, therefore, are likely to possess a common mechanism. In all of the diseases, the causative protein is proteolyzed, becomes abnormally folded and accumulates in oligomers and larger aggregates. The aggregated and possibly the monomeric expanded polyglutamine are likely to play a critical role in the pathogenesis and there is increasing evidence that the secondary structure of the protein influences its toxicity. We describe here, with special attention to huntingtin, the mechanisms of polyglutamine aggregation and the modulation of aggregation by the sequences flanking the polyglutamine. We give a comprehensive picture of the characteristics of monomeric and aggregated polyglutamine, including morphology, composition, seeding ability, secondary structure, and toxicity. The structural heterogeneity of aggregated polyglutamine may explain why polyglutamine-containing aggregates could paradoxically be either toxic or neuroprotective.

  6. Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation.

    Science.gov (United States)

    Sorolla, M Alba; Nierga, Clara; Rodríguez-Colman, M José; Reverter-Branchat, Gemma; Arenas, Alicia; Tamarit, Jordi; Ros, Joaquim; Cabiscol, Elisa

    2011-06-01

    Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Tandem reporter assay for myristoylated proteins post-translationally (TRAMPP) identifies novel substrates for post-translational myristoylation: PKCε, a case study.

    Science.gov (United States)

    Martin, Dale D O; Ahpin, Chrisselle Y; Heit, Ryan J; Perinpanayagam, Maneka A; Yap, Megan C; Veldhoen, Richard A; Goping, Ing Swie; Berthiaume, Luc G

    2012-01-01

    Myristoylation, the addition of a 14-carbon fatty acid to the N-terminal glycine of a protein, is key to protein-membrane and protein-protein interactions. Typically, myristoylation occurs cotranslationally; however, post-translational myristoylation of caspase-cleaved proteins is now emerging as a well-established protein modification and as a novel regulator of apoptosis. To identify additional post-translationally myristoylated proteins, we engineered a plasmid vector encoding for a caspase-cleavable reporter protein named tandem reporter assay for myristoylation of proteins post-translationally (TRAMPP). pTRAMPP consists of tdTomato-DEVD-"test myristoylation sequence"-enhanced green fluorescent protein (EGFP). After induction of apoptosis, the reporter protein is cleaved by caspases, which frees a new N-terminal glycine residue attached to EGFP that can be myristoylated. We used pTRAMPP in appropriately transfected cells to identify 7 post-translationally myristoylated proteins. First, we confirmed the post-translational myristoylation of two previously identified putative substrates, cytoplasmic dynein intermediate chain 2A and PKCε (ctPKCε), and identified 5 more caspase-cleaved potential substrates for myristoylation that include the antiapoptotic regulator of apoptosis, Mcl-1, and the causative agent of Huntington's disease, huntingtin protein. Further investigation revealed that post-translationally myristoylated ctPKCε localized to membranes and increased Erk signaling and degradation of the proapoptotic protein Bim, which prevented a significant loss of mitochondrial potential of 17% over nonmyristoylated ctPKCε in HeLa cells in the presence of apoptotic stimuli. Taken together, these findings suggest a possible antiapoptotic role for post-translationally myristoylated caspase-cleaved ctPKCε.

  8. Calretinin: from a simple Ca2+ buffer to a multifunctional protein implicated in many biological processes

    Directory of Open Access Journals (Sweden)

    Beat eSchwaller

    2014-02-01

    Full Text Available The hexa-EF-hand Ca2+-binding protein calretinin (CR is predominantly expressed in specific neurons of the central and peripheral nervous system. However, CR expression is also observed in non-neuronal cells, e.g. during embryonic development and in mesothelioma cells. Of the 6 EF-hand domains, 5 are functional; the first 4 domains form 2 pairs showing high cooperativity within a pair that results in non-linear modulation of intracellular Ca2+ signals by CR. EF-hand domain 5 has a low affinity and represents the identified interaction site with CR-binding partners present in mouse cerebellar granule cells. CR binding to other targets including the pore-forming α1 subunit of the Ca2+ channel CaV2.1, as well as to huntingtin indicates additional Ca2+ sensor functions besides the well-known Ca2+-buffering functions. The absence of CR in cerebellar granule cells of CR-/- mice results in increased excitability and altered firing of Purkinje cells and promotes cerebellar 160-Hz oscillations impairing motor coordination. The putative role of CR in neuroprotection is still highly discussed. Altogether, CR emerges as a multi-functional protein also associated with development, i.e. cell proliferation, differentiation and cell death.

  9. Biallelic mutations in huntington disease: A new case with just one affected parent, review of the literature and terminology.

    Science.gov (United States)

    Uhlmann, Wendy R; Peñaherrera, Maria S; Robinson, Wendy P; Milunsky, Jeff M; Nicholson, Jane M; Albin, Roger L

    2015-05-01

    Patients with biallelic mutations for Huntington disease (HD) are rare. We present a 46-year-old female with two expanded Huntingtin (HTT) alleles with just one known affected parent. This is the first reported patient with molecular studies performed to exclude HTT uniparental disomy (UPD). The proband had biparental inheritance of HTT alleles (42/44 CAG repeats). Given the negative UPD results, the proband's unaffected mother either had a reduced penetrance allele that expanded into the full mutation range during transmission to our patient or an unknown full HTT mutation and died before symptom onset, unlikely given no family history of HD and asymptomatic at age 59. We made the novel observation in our literature review that most patients with biallelic HD did not have two full HTT mutations. Most had one HTT allele that was in the intermediate or reduced penetrance ranges or 40 CAG repeats, the lowest limit of the full mutation range. Although the number of patients is small, when an allele in these size ranges was present, generally the age of HD onset was in the 50s. If the second HTT allele had >45 repeats, then onset was typically 20s-30s. While similar ages of onset have been reported for patients with one or two HTT mutations, patients with biallelic mutations may have later onset if an expanded HTT allele has ≤40 CAG repeats. Finally, we propose that "biallelic mutations" or "compound heterozygosity" are more accurate descriptive terms than "homozygosity" when there are two non-identical expanded HTT alleles.

  10. Orphan drugs in development for Huntington's disease: challenges and progress

    Directory of Open Access Journals (Sweden)

    Burgunder JM

    2015-02-01

    Full Text Available Jean-Marc Burgunder1–4 1Swiss Huntington’s Disease Centre, Department of Neurology, University of Bern, Bern, Switzerland; 2Department of Neurology, West China Hospital, Sichuan University, Chengdu, 3Department of Neurology, Xiangya Hospital, Central South University, Changsha, 4Department of Neurology, Sun Yat-sen University, Guangzhou, People’s Republic of China Abstract: Huntington’s disease is a monogenic disorder encompassing a variable phenotype with progressive cognitive, psychiatric, and movement disorders. Knowledge of the mechanisms involved in this disorder has made substantial advances since the discovery of the gene mutation. The dynamic mutation is the expansion of a CAG (cytosine-adenine-guanine repeat in the huntingtin (HTT gene, which is transcribed into an abnormal protein with an elongated polyglutamine tract. Polyglutamine HTT accumulates and is changed in its function in multifaceted ways related to the numerous roles of the normal protein. The protein is expressed in numerous areas of the brain and also in other organs. The major brain region involved in the disease process is the striatum, but it is clear that other systems are involved as well. This accumulated knowledge has now led to the development of treatment strategies based on specific molecular pathways for symptomatic and disease course-modifying treatment. The most proximal way to handle the disturbed protein is to hinder the gene transcription, translation, and/or to increase protein clearance. Other mechanisms now being approached include modulation of energy and intracellular signaling, induction of factors potentially leading to neuroprotection, as well as modulation of glial function. Several clinical trials based on these approaches are now under way, and it is becoming clear that a future disease-modifying therapy will be a combination of several approaches harmonized with symptomatic treatments. In this review, some of the most promising and

  11. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers....... The biophysical and structural investigations of PPIs consequently demand hybrid approaches, implementing orthogonal methods and strategies for global data analysis. Currently, impressive developments in hardware and software within several methodologies define a new era for the biostructural community. Data can...

  12. A unique four-hub protein cluster associates to glioblastoma progression.

    Directory of Open Access Journals (Sweden)

    Pasquale Simeone

    Full Text Available Gliomas are the most frequent brain tumors. Among them, glioblastomas are malignant and largely resistant to available treatments. Histopathology is the gold standard for classification and grading of brain tumors. However, brain tumor heterogeneity is remarkable and histopathology procedures for glioma classification remain unsatisfactory for predicting disease course as well as response to treatment. Proteins that tightly associate with cancer differentiation and progression, can bear important prognostic information. Here, we describe the identification of protein clusters differentially expressed in high-grade versus low-grade gliomas. Tissue samples from 25 high-grade tumors, 10 low-grade tumors and 5 normal brain cortices were analyzed by 2D-PAGE and proteomic profiling by mass spectrometry. This led to identify 48 differentially expressed protein markers between tumors and normal samples. Protein clustering by multivariate analyses (PCA and PLS-DA provided discrimination between pathological samples to an unprecedented extent, and revealed a unique network of deranged proteins. We discovered a novel glioblastoma control module centered on four major network hubs: Huntingtin, HNF4α, c-Myc and 14-3-3ζ. Immunohistochemistry, western blotting and unbiased proteome-wide meta-analysis revealed altered expression of this glioblastoma control module in human glioma samples as compared with normal controls. Moreover, the four-hub network was found to cross-talk with both p53 and EGFR pathways. In summary, the findings of this study indicate the existence of a unifying signaling module controlling glioblastoma pathogenesis and malignant progression, and suggest novel targets for development of diagnostic and therapeutic procedures.

  13. Metabolic disruption identified in the Huntington’s disease transgenic sheep model

    OpenAIRE

    Handley, Renee. R.; Reid, Suzanne J; Stefano Patassini; Rudiger, Skye R.; Vladimir Obolonkin; McLaughlan, Clive J.; Jacobsen, Jessie C.; Gusella, James F.; MacDonald, Marcy E.; Waldvogel, Henry J.; C Simon Bawden; Faull, Richard L. M.; Snell, Russell G.

    2016-01-01

    Huntington’s disease (HD) is a dominantly inherited, progressive neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of HTT, encoding huntingtin. There are no therapies that can delay the progression of this devastating disease. One feature of HD that may play a critical role in its pathogenesis is metabolic disruption. Consequently, we undertook a comparative study of metabolites in our transgenic sheep model of HD (OVT73). This model does not display overt symptoms of ...

  14. Protein Condensation

    Science.gov (United States)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  15. The unfolded protein response and its potential role in Huntington's disease elucidated by a systems biology approach [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Ravi Kiran Reddy Kalathur

    2016-03-01

    Full Text Available Huntington ́s disease (HD is a progressive, neurodegenerative disease with a fatal outcome. Although the disease-causing gene (huntingtin has been known for over 20 years, the exact mechanisms leading to neuronal cell death are still controversial. One potential mechanism contributing to the massive loss of neurons observed in the brain of HD patients could be the unfolded protein response (UPR activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER. As an adaptive response to counter-balance accumulation of un- or misfolded proteins, the UPR upregulates transcription of chaperones, temporarily attenuates new translation, and activates protein degradation via the proteasome. However, persistent ER stress and an activated UPR can also cause apoptotic cell death. Although different studies have indicated a role for the UPR in HD, the evidence remains inconclusive. Here, we present extensive bioinformatic analyses that revealed UPR activation in different experimental HD models based on transcriptomic data. Accordingly, we have identified 53 genes, including RAB5A, HMGB1, CTNNB1, DNM1, TUBB, TSG101, EEF2, DYNC1H1, SLC12A5, ATG5, AKT1, CASP7 and SYVN1 that provide a potential link between UPR and HD. To further elucidate the potential role of UPR as a disease-relevant process, we examined its connection to apoptosis based on molecular interaction data, and identified a set of 40 genes including ADD1, HSP90B1, IKBKB, IKBKG, RPS3A and LMNB1, which seem to be at the crossroads between these two important cellular processes. Remarkably, we also found strong correlation of UPR gene expression with the length of the polyglutamine tract of Huntingtin, which is a critical determinant of age of disease onset in human HD patients pointing to the UPR as a promising target for therapeutic intervention. The study is complemented by a newly developed web-portal called UPR-HD (http://uprhd.sysbiolab.eu that enables visualization and

  16. Protein C

    Science.gov (United States)

    ... have an unexplained blood clot, or a family history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with the function of this protein may cause blood clots to ...

  17. Protein S

    Science.gov (United States)

    ... have an unexplained blood clot, or a family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with the function of this protein may cause blood clots to ...

  18. Characterization of Behavioral, Neuropathological, Brain Metabolic and Key Molecular Changes in zQ175 Knock-In Mouse Model of Huntington's Disease.

    Directory of Open Access Journals (Sweden)

    Qi Peng

    Full Text Available Huntington's disease (HD is caused by an expansion of the trinucleotide poly (CAG tract located in exon 1 of the huntingtin (Htt gene leading to progressive neurodegeneration in selected brain regions, and associated functional impairments in motor, cognitive, and psychiatric domains. Since the discovery of the gene mutation that causes the disease, mouse models have been developed by different strategies. Recently, a new model, the zQ175 knock-in (KI line, was developed in an attempt to have the Htt gene in a context and causing a phenotype that more closely mimics HD in humans. The behavioral phenotype was characterized across the independent laboratories and important features reminiscent of human HD are observed in zQ175 mice. In the current study, we characterized the zQ175 model housed in an academic laboratory under reversed dark-light cycle, including motor function, in vivo longitudinal structural MRI imaging for brain volume, MRS for striatal metabolites, neuropathology, as well as a panel of key disease marker proteins in the striatum at different ages. Our results suggest that homozygous zQ175 mice exhibited significant brain atrophy before the motor deficits and brain metabolite changes. Altered striatal medium spiny neuronal marker, postsynaptic marker protein and complement component C1qC also characterized zQ175 mice. Our results confirmed that the zQ175 KI model is valuable in understanding of HD-like pathophysiology and evaluation of potential therapeutics. Our data also provide suggestions to select appropriate outcome measurements in preclinical studies using the zQ175 mice.

  19. A majority of Huntington's disease patients may be treatable by individualized allele-specific RNA interference.

    Science.gov (United States)

    Lombardi, Maria Stella; Jaspers, Leonie; Spronkmans, Christine; Gellera, Cinzia; Taroni, Franco; Di Maria, Emilio; Donato, Stefano Di; Kaemmerer, William F

    2009-06-01

    Use of RNA interference to reduce huntingtin protein (htt) expression in affected brain regions may provide an effective treatment for Huntington disease (HD), but it remains uncertain whether suppression of both wild-type and mutant alleles in a heterozygous patient will provide more benefit than harm. Previous research has shown suppression of just the mutant allele is achievable using siRNA targeted to regions of HD mRNA containing single nucleotide polymorphisms (SNPs). To determine whether more than a minority of patients may be eligible for an allele-specific therapy, we genotyped DNA from 327 unrelated European Caucasian HD patients at 26 SNP sites in the HD gene. Over 86% of the patients were found to be heterozygous for at least one SNP among those tested. Because the sites are genetically linked, one cannot use the heterozygosity rates of the individual SNPs to predict how many sites (and corresponding allele-specific siRNA) would be needed to provide at least one treatment possibility for this percentage of patients. By computing all combinations, we found that a repertoire of allele-specific siRNA corresponding to seven sites can provide at least one allele-specific siRNA treatment option for 85.6% of our sample. Moreover, we provide evidence that allele-specific siRNA targeting these sites are readily identifiable using a high throughput screening method, and that allele-specific siRNA identified using this method indeed show selective suppression of endogenous mutant htt protein in fibroblast cells from HD patients. Therefore, allele-specific siRNA are not so rare as to be impractical to find and use therapeutically.

  20. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  1. Dysfunction of the CNS-heart axis in mouse models of Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Michal Mielcarek

    2014-08-01

    Full Text Available Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer's, Parkinson's and Huntington's disease (HD. While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM. This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor.

  2. Dysfunction of the CNS-heart axis in mouse models of Huntington's disease.

    Science.gov (United States)

    Mielcarek, Michal; Inuabasi, Linda; Bondulich, Marie K; Muller, Thomas; Osborne, Georgina F; Franklin, Sophie A; Smith, Donna L; Neueder, Andreas; Rosinski, Jim; Rattray, Ivan; Protti, Andrea; Bates, Gillian P

    2014-08-01

    Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer's, Parkinson's and Huntington's disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor.

  3. Epigenetic DNA Methylation Profiling with MSRE: A Quantitative NGS Approach Using a Parkinson's Disease Test Case

    Directory of Open Access Journals (Sweden)

    Adam G. Marsh

    2016-11-01

    Full Text Available Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS data using methyl-sensitive restriction endonucleases (MSRE. An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1,008 statistically different CpG sites (p textless 0.0025, after false discovery rate correction. A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations. Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression.

  4. Safety of striatal infusion of siRNA in a transgenic Huntington's disease mouse model

    Science.gov (United States)

    McGowan, Sarah; Mondo, Erica; Pfister, Edith; Mick, Eric; Friedline, Randall H.; Kim, Jason K.; Sapp, Ellen; DiFiglia, Marian; Aronin, Neil

    2016-01-01

    Background The immune system In Huntington's disease (HD) is activated and may overreact to some therapies. RNA interference using siRNA lowers mutant huntingtin (mHTT) protein but could increase immune responses. Objective To examine the innate immune response following siRNA infusion into the striatum of wild-type (WT) and HD transgenic (YAC128) mice. Methods siRNAs (2′-O-methyl phosphorothioated) were infused unilaterally into striatum of four month-old WT and YAC128 mice for 28 days. Microglia number and morphology (resting (normal), activated, dystrophic), cytokine levels, and DARPP32-positive neurons were measured in striatum immediately or 14 days post-infusion. Controls included contralateral untreated striatum, and PBS and sham treated striata. Results The striata of untreated YAC128 mice had significantly fewer resting microglia and more dystrophic microglia than WT mice, but no difference from WT in the proportion of activated microglia or total number of microglia. siRNA infusion increased the total number of microglia in YAC128 mice compared to PBS treated and untreated striata and increased the proportion of activated microglia in WT and YAC128 mice compared to untreated striata and sham treated groups. Cytokine levels were low and siRNA infusion resulted in only modest changes in those levels. siRNA infusion did not change the number of DARPP32-positive neurons. Conclusion Findings suggest that siRNA infusion may be a safe method for lowering mHTT levels in the striatum in young animals, since treatment does not produce a robust cytokine response or cause neurotoxicity. The potential long-term effects of a sustained increase in total and activated microglia after siRNA infusion in HD mice need to be explored. PMID:26444021

  5. Protein Structure

    Science.gov (United States)

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  6. Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

    Directory of Open Access Journals (Sweden)

    Ricardo Mouro Pinto

    2013-10-01

    Full Text Available The Huntington's disease gene (HTT CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111 than on a 129 background (129.Hdh(Q111 . Linkage mapping in (B6x129.Hdh(Q111 F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111 somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3 complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3. The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest

  7. Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

    Science.gov (United States)

    Pinto, Ricardo Mouro; Dragileva, Ella; Kirby, Andrew; Lloret, Alejandro; Lopez, Edith; St Claire, Jason; Panigrahi, Gagan B; Hou, Caixia; Holloway, Kim; Gillis, Tammy; Guide, Jolene R; Cohen, Paula E; Li, Guo-Min; Pearson, Christopher E; Daly, Mark J; Wheeler, Vanessa C

    2013-10-01

    The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1

  8. The role of tau in the pathological process and clinical expression of Huntington's disease.

    Science.gov (United States)

    Vuono, Romina; Winder-Rhodes, Sophie; de Silva, Rohan; Cisbani, Giulia; Drouin-Ouellet, Janelle; Spillantini, Maria G; Cicchetti, Francesca; Barker, Roger A

    2015-07-01

    Huntington's disease is a neurodegenerative disorder caused by an abnormal CAG repeat expansion within exon 1 of the huntingtin gene HTT. While several genetic modifiers, distinct from the Huntington's disease locus itself, have been identified as being linked to the clinical expression and progression of Huntington's disease, the exact molecular mechanisms driving its pathogenic cascade and clinical features, especially the dementia, are not fully understood. Recently the microtubule associated protein tau, MAPT, which is associated with several neurodegenerative disorders, has been implicated in Huntington's disease. We explored this association in more detail at the neuropathological, genetic and clinical level. We first investigated tau pathology by looking for the presence of hyperphosphorylated tau aggregates, co-localization of tau with mutant HTT and its oligomeric intermediates in post-mortem brain samples from patients with Huntington's disease (n = 16) compared to cases with a known tauopathy and healthy controls. Next, we undertook a genotype-phenotype analysis of a large cohort of patients with Huntington's disease (n = 960) with a particular focus on cognitive decline. We report not only on the tau pathology in the Huntington's disease brain but also the association between genetic variation in tau gene and the clinical expression and progression of the disease. We found extensive pathological inclusions containing abnormally phosphorylated tau protein that co-localized in some instances with mutant HTT. We confirmed this related to the disease process rather than age, by showing it is also present in two patients with young-onset Huntington's disease (26 and 40 years old at death). In addition we demonstrate that tau oligomers (suggested to be the most likely neurotoxic tau entity) are present in the Huntington's disease brains. Finally we highlight the clinical significance of this pathology by demonstrating that the MAPT haplotypes affect the rate

  9. Progress in studies of gene therapy for Huntington's disease%亨廷顿病基因治疗研究进展

    Institute of Scientific and Technical Information of China (English)

    金范莹; 张宝荣

    2012-01-01

    亨廷顿病是一种以运动、认知和精神障碍为主要表现的遗传性中枢神经系统变性疾病,其致病基因IT-15突变可引起胞嘧啶-腺嘌呤-鸟嘌呤(CAG)三核苷酸重复序列异常扩增,导致所编码的亨廷顿蛋白构象变化并产牛神经毒性作用.亨廷顿病的基因治疗目前尚处于临床前阶段,主要包括摹因沉默、诱导突变亨廷顿蛋白清除、导入神经营养冈子基因,以及纠正突变型亨廷顿蛋白的毒性作用所致的基因转录、信号转导和线粒体代谢紊乱等.本文尝试对亨廷顿病的基因治疗研究进展简要叙述.%Huntington's disease (HD) is a kind of inherited neurodegenerative disorder characterized by movement problems, cognitive decline and psychiatry disturbance. HD is caused by mutation in gene IT-15 involving the expansion of a trinucleotide (CAG) repeat encoding glutamine, which leads to abnormal conformation of huntingtin (Htt) protein and finally emerge cytotoxic functions. Currently, HD remains a fatal untreatable disease. Gene therapy for HD discussed in this review is under preclinical studies. Silencing of mutant IT-15 via RNA interference (RNAi) or antisense oligonucleotide (ASO) has shown some effectiveness in mouse model studies. Increasing the clearance of mutant Htt protein could be achieved by viral-mediated delivery of anti-Htt intrabodies (lAbs) or induction of autophagy, and beneficial results have been observed. Ectopic expression of neurotrophic factors, such as nerve growth factor (NGF) and brain - derived neurotrophic factor (BDNF), mediated either by viral vectors or transplantation of genetically modified cells, has also been proved to be effective. Other gene - modifying methods aiming at correction of transcriptional dysregulation by histone modification, activation of endogenous neural stem cells, and normalization of calcium signaling and mitochondrial function, are also under intensive research. Gene therapy for Huntington

  10. Whey Protein

    Science.gov (United States)

    ... Fraction de Lactosérum, Fraction de Petit-Lait, Goat Milk Whey, Goat Whey, Isolat de Protéine de Lactosérum, Isolat ... Lactosérum de Lait de Chèvre, MBP, Milk Protein, Milk Protein Isolate, Mineral Whey Concentrate, Proteínas del Suero de la Leche, Protéine ...

  11. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  12. The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease

    Science.gov (United States)

    Sameni, Sara; Syed, Adeela; Marsh, J. Lawrence; Digman, Michelle A.

    2016-10-01

    Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening.

  13. Huntington's Disease and Striatal Signaling

    Directory of Open Access Journals (Sweden)

    Emmanuel eRoze

    2011-08-01

    Full Text Available Huntington’s Disease (HD is the most frequent neurodegenerative disease caused by an expansion of polyglutamines (CAG. The main clinical manifestations of HD are chorea, cognitive impairment and psychiatric disorders. The transmission of HD is autosomal dominant with a complete penetrance. HD has a single genetic cause, a well-defined neuropathology, and informative pre-manifest genetic testing of the disease is available. Striatal atrophy begins as early as 15 years before disease onset and continues throughout the period of manifest illness. Therefore, patients could theoretically benefit from therapy at early stages of the disease. One important characteristic of HD is the striatal vulnerability to neurodegeneration, despite similar expression of the protein in other brain areas. Aggregation of the mutated Huntingtin (HTT, impaired axonal transport, excitotoxicity, transcriptional dysregulation as well as mitochondrial dysfunction and energy deficits, are all part of the cellular events that underlie neuronal dysfunction and striatal death. Among these non-exclusive mechanisms, an alteration of striatal signaling is thought to orchestrate the downstream events involved in the cascade of striatal dysfunction.

  14. Huntington's Disease and Striatal Signaling.

    Science.gov (United States)

    Roze, Emmanuel; Cahill, Emma; Martin, Elodie; Bonnet, Cecilia; Vanhoutte, Peter; Betuing, Sandrine; Caboche, Jocelyne

    2011-01-01

    Huntington's Disease (HD) is the most frequent neurodegenerative disease caused by an expansion of polyglutamines (CAG). The main clinical manifestations of HD are chorea, cognitive impairment, and psychiatric disorders. The transmission of HD is autosomal dominant with a complete penetrance. HD has a single genetic cause, a well-defined neuropathology, and informative pre-manifest genetic testing of the disease is available. Striatal atrophy begins as early as 15 years before disease onset and continues throughout the period of manifest illness. Therefore, patients could theoretically benefit from therapy at early stages of the disease. One important characteristic of HD is the striatal vulnerability to neurodegeneration, despite similar expression of the protein in other brain areas. Aggregation of the mutated Huntingtin (HTT), impaired axonal transport, excitotoxicity, transcriptional dysregulation as well as mitochondrial dysfunction, and energy deficits, are all part of the cellular events that underlie neuronal dysfunction and striatal death. Among these non-exclusive mechanisms, an alteration of striatal signaling is thought to orchestrate the downstream events involved in the cascade of striatal dysfunction.

  15. A systematic review of the intergenerational aspects and the diverse genetic profiles of Huntington's disease.

    Science.gov (United States)

    Agostinho, L A; Dos Santos, S R; Alvarenga, R M P; Paiva, C L A

    2013-06-13

    Huntington's disease (HD) is a rare progressive and fatal neurogenetic degenerative disease, characterized by movement and personality disorders and by progressive dementia. Its prevalence varies by ethnic origin and different genetic profiles predisposing individuals to HD in each population. The prevalence of HD is 5-10 per 100,000 individuals in Caucasian populations of North America and Western Europe. It is an autosomal dominant disease associated with the expansion of CAG-type repetitive DNA sequences in the HTT gene. This gene, located on the short arm of chromosome 4, encodes the protein huntingtin. In this study, we reviewed 17 articles about HD that report data from 2400 affected individuals from various countries around the world, including Venezuela, China, Croatia, Turkey, Germany, Italy, Brazil, Spain, Taiwan, India, the Netherlands, Russia, and the USA, with a focus on genetic profiles and intergenerational expansions or contractions of expanded alleles responsible for causing HD. We discuss the genetic characteristics of HD in different populations and any atypical cases reported in these studies.

  16. The Neuropathology of Huntington´s disease: classical findings, recent developments and correlation to functional neuroanatomy.

    Science.gov (United States)

    Rüb, Udo; Vonsattel, Jean Paul V; Heinsen, Helmut; Korf, Horst-Werner

    2015-01-01

    Huntington’s disease (HD) is a severe, autosomal dominantly inherited, gradually worsening neurological disorder, the clinical features of which were first described in 1863 by Irving W. Lyon and with additional details, in 1872, by George Huntington. Progress in molecular biological research has shown that HD is caused by meiotically unstable CAG-repeats in the mutated HD gene (the so-called IT 15 gene) on chromosome 4p16.3, which encodes the mutated protein huntingtin (Htt). This monograph provides a survey of the stepwise progress in neuropathological HD research made during a time period of more than hundred years, the currently known neuropathological hallmarks of HD, as well as their pathogenic and clinical relevance. Starting with the initial descriptions of the progressive degeneration of the neostriatum (i.e., caudate nucleus and putamen) as one of the key events in HD, the worldwide practiced Vonsattel HD grading system of striatal neurodegeneration will be outlined. Correlating qualitative and quantitative neuropathological data with characteristics pertaining to the functional neuroanatomy of the human brain, subsequent chapters will highlight the latest neuropathological HD findings: the area- and layer-specifi c neuronal loss in the cerebral neo- and allocortex, the neurodegeneration of select thalamic nuclei, the affection of the cerebellar cortex and the deep cerebellar nuclei, the involvement of distinct brainstem nuclei, and the pathophysiological relevance of these pathologies for the clinical phenotype of HD. Finally, the potential pathophysiological role of axonal transport deficit

  17. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware......Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  18. Novel BAC mouse model of Huntington’s disease with 225 CAG repeats exhibits an early widespread and stable degenerative phenotype

    Science.gov (United States)

    Wegrzynowicz, Michal; Bichell, Terry Jo; Soares, Barbara D.; Loth, Meredith K.; McGlothan, Jennifer L.; Alikhan, Fatima S.; Hua, Kegang; Coughlin, Jennifer M.; Holt, Hunter K.; Jetter, Christopher S.; Mori, Susumu; Pomper, Martin G.; Osmand, Alexander P.; Guilarte, Tomás R.; Bowman, Aaron B.

    2015-01-01

    BACKGROUND Unusually large CAG repeat expansions (>60) in exon one of Huntingtin (HTT) are invariably associated with a juvenile-onset form of Huntington’s disease (HD), characterized by a more extensive and rapidly progressing neuropathology than the more prevalent adult-onset form. However, existing mouse models of HD that express the full-length Htt gene with CAG repeat lengths associated with juvenile HD (ranging between ~75 to ~150 repeats in published models) exhibit selective neurodegenerative phenotypes more consistent with adult-onset HD. OBJECTIVE To determine if a very large CAG repeat (>200) in full-length Htt elicits neurodegenerative phenotypes consistent with juvenile HD. METHODS Using a bacterial artificial chromosome (BAC) system, we generated mice expressing full-length mouse Htt with ~225 CAG repeats under control of the mouse Htt promoter. Mice were characterized using behavioral, neuropathological, biochemical and brain imaging methods. RESULTS BAC-225Q mice exhibit phenotypes consistent with a subset of features seen in juvenile-onset HD: very early motor behavior abnormalities, reduced body weight, widespread and progressive increase in Htt aggregates, gliosis, and neurodegeneration. Early striatal pathology was observed, including reactive gliosis and loss of dopamine receptors, prior to detectable volume loss. HD-related blood markers of impaired energy metabolism and systemic inflammation were also increased. Aside from an age-dependent progression of diffuse nuclear aggregates at 6 months of age to abundant neuropil aggregates at 12 months of age, other pathological and motor phenotypes showed little to no progression. CONCLUSIONS The HD phenotypes present in animals 3 to 12 months of age make the BAC-225Q mice a unique and stable model of full-length mutant Htt associated phenotypes, including body weight loss, behavioral impairment and HD-like neurodegenerative phenotypes characteristic of juvenile-onset HD and/or late-stage adult

  19. Protein Crystallization

    Science.gov (United States)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  20. Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway.

    Science.gov (United States)

    Szlachcic, Wojciech J; Switonski, Pawel M; Krzyzosiak, Wlodzimierz J; Figlerowicz, Marek; Figiel, Maciej

    2015-09-01

    Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life.

  1. Protein inference: A protein quantification perspective.

    Science.gov (United States)

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  2. Amygdala Function and 5-HTT Gene Variants in Adolescent Anxiety and Major Depressive Disorder

    Science.gov (United States)

    Lau, Jennifer Y. F.; Goldman, David; Buzas, Beata; Fromm, Stephen J.; Guyer, Amanda E.; Hodgkinson, Colin; Monk, Christopher S.; Nelson, Eric E.; Shen, Pei-Hong; Pine, Daniel S.; Ernst, Monique

    2009-01-01

    Background Associations between a functional polymorphism in the serotonin transporter gene and amygdala activation have been found in healthy, depressed, and anxious adults. This study explored these gene–brain associations in adolescents by examining predictive effects of serotonin transporter gene variants (S and LG allele carriers vs. LA allele homozygotes) and their interaction with diagnosis (healthy vs. patients) on amygdala responses to emotional faces. Methods Functional magnetic resonance data were collected from 33 healthy adolescents (mean age: 13.71, 55% female) and 31 medication-free adolescents with current anxiety or depressive disorders (or both; mean age: 13.58, 56% female) while viewing fearful, angry, happy, and neutral facial expressions under varying attention states. Results A significant three-way genotype-by-diagnosis-by-face-emotion interaction characterized right amygdala activity while subjects monitored internal fear levels. This interaction was decomposed to map differential gene–brain associations in healthy and affected adolescents. First, consistent with healthy adult data, healthy adolescents with at least one copy of the S or LG allele showed stronger amygdala responses to fearful faces than healthy adolescents without these alleles. Second, patients with two copies of the LA allele exhibited greater amygdala responses to fearful faces relative to patients with S or LG alleles. Third, although weaker, genotype differences on amygdala responses in patients extended to happy faces. All effects were restricted to the fear-monitoring attention state. Conclusions S/LG alleles in healthy adolescents, as in healthy adults, predict enhanced amygdala activation to fearful faces. Contrary findings of increased activation in patients with LALA relative to the S or LG alleles require further exploration. PMID:18950748

  3. Clinical features of Chinese patients with Huntington's disease carrying CAG repeats beyond 60 within HTT gene.

    Science.gov (United States)

    Liu, Z-J; Sun, Y-M; Ni, W; Dong, Y; Shi, S-S; Wu, Z-Y

    2014-02-01

    Patients with Huntington's disease (HD) carrying CAG repeats beyond 60 are less frequently seen and clinical features of them have been rarely reported. We identified four unrelated patients carrying CAG repeats beyond 60 (84.0 ± 13.76, ranging from 74 to 104) from 119 Chinese HD patients via direct sequencing. These four were all early onset with a mean age at presenting symptom of 9.8 ± 1.71 years. Paternal transmission was found in three of them and the fourth was apparently sporadic. In addition, they had atypical onset symptoms including epilepsy, intellectual decline, tics and walking instability, which might lead the clinicians to make the wrong diagnosis in the early stage of disease. Our work explores clinical features of Chinese HD patients with an expanded CAG repeat over 60 and may help the clinicians make a correct diagnosis in the early stage of disease.

  4. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  5. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein i

  6. MicroRNA-22 (miR-22 overexpression is neuroprotective via general anti-apoptotic effects and may also target specific Huntington's disease-related mechanisms.

    Directory of Open Access Journals (Sweden)

    Ana Jovicic

    Full Text Available BACKGROUND: Whereas many causes and mechanisms of neurodegenerative diseases have been identified, very few therapeutic strategies have emerged in parallel. One possible explanation is that successful treatment strategy may require simultaneous targeting of more than one molecule of pathway. A new therapeutic approach to have emerged recently is the engagement of microRNAs (miRNAs, which affords the opportunity to target multiple cellular pathways simultaneously using a single sequence. METHODOLOGY/PRINCIPAL FINDINGS: We identified miR-22 as a potentially neuroprotective miRNA based on its predicted regulation of several targets implicated in Huntington's disease (histone deacetylase 4 (HDAC4, REST corepresor 1 (Rcor1 and regulator of G-protein signaling 2 (Rgs2 and its diminished expression in Huntington's and Alzheimer's disease brains. We then tested the hypothesis that increasing cellular levels of miRNA-22 would achieve neuroprotection in in vitro models of neurodegeneration. As predicted, overexpression of miR-22 inhibited neurodegeneration in primary striatal and cortical cultures exposed to a mutated human huntingtin fragment (Htt171-82Q. Overexpression of miR-22 also decreased neurodegeneration in primary neuronal cultures exposed to 3-nitropropionic acid (3-NP, a mitochondrial complex II/III inhibitor. In addition, miR-22 improved neuronal viability in an in vitro model of brain aging. The mechanisms underlying the effects of miR-22 included a reduction in caspase activation, consistent with miR-22's targeting the pro-apoptotic activities of mitogen-activated protein kinase 14/p38 (MAPK14/p38 and tumor protein p53-inducible nuclear protein 1 (Tp53inp1. Moreover, HD-specific effects comprised not only targeting HDAC4, Rcor1 and Rgs2 mRNAs, but also decreasing focal accumulation of mutant Htt-positive foci, which occurred via an unknown mechanism. CONCLUSIONS: These data show that miR-22 has multipartite anti-neurodegenerative activities

  7. Update on Huntington's disease: advances in care and emerging therapeutic options.

    Science.gov (United States)

    Zielonka, Daniel; Mielcarek, Michal; Landwehrmeyer, G Bernhard

    2015-03-01

    Huntington's disease (HD) is the most common hereditary neurodegenerative disorder. Despite the fact that both the gene and the mutation causing this monogenetic disorder were identified more than 20 years ago, disease-modifying therapies for HD have not yet been established. While intense preclinical research and large cohort studies in HD have laid foundations for tangible improvements in understanding HD and caring for HD patients, identifying targets for therapeutic interventions and developing novel therapeutic modalities (new chemical entities and advanced therapies using DNA and RNA molecules as therapeutic agents) continues to be an ongoing process. The authors review recent achievements in HD research and focus on approaches towards disease-modifying therapies, ranging from huntingtin-lowering strategies to improving huntingtin clearance that may be promoted by posttranslational HTT modifications. The nature and number of upcoming clinical studies/trials in HD is a reason for hope for HD patients and their families. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Wheel running from a juvenile age delays onset of specific motor deficits but does not alter protein aggregate density in a mouse model of Huntington's disease

    Directory of Open Access Journals (Sweden)

    Spires Tara L

    2008-04-01

    Full Text Available Abstract Background Huntington's disease (HD is a neurodegenerative disorder predominantly affecting the cerebral cortex and striatum. Transgenic mice (R6/1 line, expressing a CAG repeat encoding an expanded polyglutamine tract in the N-terminus of the huntingtin protein, closely model HD. We have previously shown that environmental enrichment of these HD mice delays the onset of motor deficits. Furthermore, wheel running initiated in adulthood ameliorates the rear-paw clasping motor sign, but not an accelerating rotarod deficit. Results We have now examined the effects of enhanced physical activity via wheel running, commenced at a juvenile age (4 weeks, with respect to the onset of various behavioral deficits and their neuropathological correlates in R6/1 HD mice. HD mice housed post-weaning with running wheels only, to enhance voluntary physical exercise, have delayed onset of a motor co-ordination deficit on the static horizontal rod, as well as rear-paw clasping, although the accelerating rotarod deficit remains unaffected. Both wheel running and environmental enrichment rescued HD-induced abnormal habituation of locomotor activity and exploratory behavior in the open field. We have found that neither environment enrichment nor wheel running ameliorates the shrinkage of the striatum and anterior cingulate cortex (ACC in HD mice, nor the overall decrease in brain weight, measured at 9 months of age. At this age, the density of ubiquitinated protein aggregates in the striatum and ACC is also not significantly ameliorated by environmental enrichment or wheel running. Conclusion These results indicate that enhanced voluntary physical activity, commenced at an early presymptomatic stage, contributes to the positive effects of environmental enrichment. However, sensory and cognitive stimulation, as well as motor stimulation not associated with running, may constitute major components of the therapeutic benefits associated with enrichment

  9. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  10. Regulation of mitochondrial morphology and cell cycle by microRNA-214 targeting Mitofusin2.

    Science.gov (United States)

    Bucha, Sudha; Mukhopadhyay, Debashis; Bhattacharyya, Nitai Pada

    2015-10-02

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the increase in CAG repeats beyond 36 at the exon1 of the gene Huntingtin (HTT). Among the various dysfunctions of biological processes in HD, transcription deregulation due to abnormalities in actions of transcription factors has been considered to be one of the important pathological conditions. In addition, deregulation of microRNA (miRNA) expression has been described in HD. Earlier, expression of microRNA-214 (miR-214) has been shown to increase in HD cell models and target HTT gene; the expression of the later being inversely correlated to that of miR-214. In the present communication, we observed that the expressions of several HTT co-expressed genes are modulated by exogenous expression of miR-214 or by its mutant. Among several HTT co-expressed genes, MFN2 was shown to be the direct target of miR-214. Exogenous expression of miR-214, repressed the expression of MFN2, increased the distribution of fragmented mitochondria and altered the distribution of cells in different phases of cell cycle. In summary, we have shown that increased expression of miR-214 observed in HD cell model could target MFN2, altered mitochondrial morphology and deregulated cell cycle. Inhibition of miR-214 could be a possible target of intervention in HD pathogenesis.

  11. Effects of Pin1 loss in HdhQ111 knock-in mice

    Directory of Open Access Journals (Sweden)

    Elena eAgostoni

    2016-05-01

    Full Text Available Huntington’s disease (HD is a fatal, dominantly inherited, neurodegenerative disorder due to a pathological expansion of the CAG repeat in the coding region of the HTT gene. In the quest for understanding the molecular basis of neurodegeneration, we have previously demonstrated that the prolyl isomerase Pin1 plays a crucial role in mediating p53-dependent apoptosis triggered by mutant huntingtin (mHtt in vitro. To assess the effects of the lack of Pin1 in vivo, we have bred Pin1 knock-out mice with HdhQ111 knock-in mice, a genetically precise model of HD. We show that Pin1 genetic ablation modifies a portion of HdhQ111 phenotypes in a time-dependent fashion. As an early event, Pin1 activity reduces the DNA damage response. In midlife mice, by taking advantage of next-generation sequencing technology, we show that Pin1 activity modulates a portion of the alterations triggered by mHtt, extending the role of Pin1 to two additional HdhQ111 phenotypes: the unbalance in the synthesis/concentration of hormones, as well as the alteration of Wnt/β-catenin signaling. In aging animals, Pin1 significantly increases the number of mHtt-positive nuclear inclusions while it reduces gliosis. In summary, this work provides further support for a role of Pin1 in HD pathogenesis.

  12. Chinese patients with Huntington's disease initially presenting with spinocerebellar ataxia.

    Science.gov (United States)

    Dong, Y; Sun, Y-M; Liu, Z-J; Ni, W; Shi, S-S; Wu, Z-Y

    2013-04-01

    Recent studies have described Huntington's disease (HD) patients with atypical onset of ataxia. Symptoms in these patients can overlap with those of spinocerebellar ataxia (SCA). We retrospectively examined clinical data for 82 HD probands and found 7 had initially been clinically diagnosed as SCA cases. Clinical features in these patients were further investigated and the number of CAG repeats in the huntingtin (HTT) gene was determined by direct sequencing. Genetic screenings for SCAs in the 7 patients were all negative. By contrast, HTT was heterozygous in each patient. The distribution of CAG number in the 7 patients was statistically the same as that in the other 75 patients. Each of 7 HD patients had presented with atypical onset of ataxia. The mean time from onset to HTT genetic testing was 5.6 ± 5.52 years. Three of the patients developed chorea, but the others did not. Our observations confirm the clinical heterogeneity of HD in Han Chinese. Based on these findings, testing for HTT expansions should be considered for clinically diagnosed SCA patients who test negatively in genetic screening of SCA genes.

  13. Molecular principles of protein stability and protein-protein interactions

    OpenAIRE

    Lendel, Christofer

    2005-01-01

    Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z ...

  14. Small heat shock proteins, protein degradation and protein aggregation diseases

    NARCIS (Netherlands)

    Vos, Michel J.; Zijlstra, Marianne P.; Carra, Serena; Sibon, Ody C. M.; Kampinga, Harm H.

    Small heat shock proteins have been characterized in vitro as ATP-independent molecular chaperones that can prevent aggregation of un- or misfolded proteins and assist in their refolding with the help of ATP-dependent chaperone machines (e. g., the Hsp70 proteins). Comparison of the functionality of

  15. EDITORIAL: Precision proteins Precision proteins

    Science.gov (United States)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  16. Fusion-protein-assisted protein crystallization.

    Science.gov (United States)

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

  17. Scaffolds for blocking protein-protein interactions.

    Science.gov (United States)

    Hershberger, Stefan J; Lee, Song-Gil; Chmielewski, Jean

    2007-01-01

    Due to the pivotal roles that protein-protein interactions play in a plethora of biological processes, the design of therapeutic agents targeting these interactions has become an attractive and important area of research. The development of such agents is faced with a variety of challenges. Nevertheless, considerable progress has been made in the design of proteomimetics capable of disrupting protein-protein interactions. Those inhibitors based on molecular scaffold designs hold considerable interest because of the ease of variation in regard to their displayed functionality. In particular, protein surface mimetics, alpha-helical mimetics, beta-sheet/beta-strand mimetics, as well as beta-turn mimetics have successfully modulated protein-protein interactions involved in such diseases as cancer and HIV. In this review, current progress in the development of molecular scaffolds designed for the disruption of protein-protein interactions will be discussed with an emphasis on those active against biological targets.

  18. Protein docking prediction using predicted protein-protein interface

    Directory of Open Access Journals (Sweden)

    Li Bin

    2012-01-01

    Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  19. Protein Crystal Based Nanomaterials

    Science.gov (United States)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  20. Shotgun protein sequencing.

    Energy Technology Data Exchange (ETDEWEB)

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  1. Protein folding, protein homeostasis, and cancer

    Institute of Scientific and Technical Information of China (English)

    John H. Van Drie

    2011-01-01

    Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery.

  2. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  3. Protein-losing enteropathy

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  4. Protein and Heart Health

    Science.gov (United States)

    ... It Works Healthy Workplace Food and Beverage Toolkit Protein and Heart Health Updated:May 5,2015 Protein ... said. What’s the harm in getting too much protein? The main problem is that often the extra ...

  5. Identification of elevated urea as a severe, ubiquitous metabolic defect in the brain of patients with Huntington's disease.

    Science.gov (United States)

    Patassini, Stefano; Begley, Paul; Reid, Suzanne J; Xu, Jingshu; Church, Stephanie J; Curtis, Maurice; Dragunow, Mike; Waldvogel, Henry J; Unwin, Richard D; Snell, Russell G; Faull, Richard L M; Cooper, Garth J S

    Huntington's disease (HD) is a neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein through the lengthening of a polyglutamine tract and initiates a cascade that ultimately leads to dementia and premature death. However, neurodegeneration typically manifests in HD only in middle age, and processes linking the causative mutation to brain disease are poorly understood. Here, our objective was to elucidate further the processes that cause neurodegeneration in HD, by measuring levels of metabolites in brain regions known to undergo varying degrees of damage. We applied gas-chromatography/mass spectrometry-based metabolomics in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine controls. Unexpectedly, a single major abnormality was evident in all eleven brain regions studied across the forebrain, midbrain and hindbrain, namely marked elevation of urea, a metabolite formed in the urea cycle by arginase-mediated cleavage of arginine. Urea cycle activity localizes primarily in the liver, where it functions to incorporate protein-derived amine-nitrogen into urea for recycling or urinary excretion. It also occurs in other cell-types, but systemic over-production of urea is not known in HD. These findings are consistent with impaired local urea regulation in brain, by up-regulation of synthesis and/or defective clearance. We hypothesize that defective brain urea metabolism could play a substantive role in the pathogenesis of neurodegeneration, perhaps via defects in osmoregulation or nitrogen metabolism. Brain urea metabolism is therefore a target for generating novel monitoring/imaging strategies and/or therapeutic interventions aimed at ameliorating the impact of HD in patients.

  6. Conductometric monitoring of protein-protein interactions.

    Science.gov (United States)

    Spera, Rosanna; Festa, Fernanda; Bragazzi, Nicola L; Pechkova, Eugenia; LaBaer, Joshua; Nicolini, Claudio

    2013-12-06

    Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.

  7. Protein Structure Prediction by Protein Threading

    Science.gov (United States)

    Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

    The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

  8. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  9. Prediction of Protein-Protein Interactions Using Protein Signature Profiling

    Institute of Scientific and Technical Information of China (English)

    Mahmood A. Mahdavi; Yen-Han Lin

    2007-01-01

    Protein domains are conserved and functionally independent structures that play an important role in interactions among related proteins. Domain-domain inter- actions have been recently used to predict protein-protein interactions (PPI). In general, the interaction probability of a pair of domains is scored using a trained scoring function. Satisfying a threshold, the protein pairs carrying those domains are regarded as "interacting". In this study, the signature contents of proteins were utilized to predict PPI pairs in Saccharomyces cerevisiae, Caenorhabditis ele- gans, and Homo sapiens. Similarity between protein signature patterns was scored and PPI predictions were drawn based on the binary similarity scoring function. Results show that the true positive rate of prediction by the proposed approach is approximately 32% higher than that using the maximum likelihood estimation method when compared with a test set, resulting in 22% increase in the area un- der the receiver operating characteristic (ROC) curve. When proteins containing one or two signatures were removed, the sensitivity of the predicted PPI pairs in- creased significantly. The predicted PPI pairs are on average 11 times more likely to interact than the random selection at a confidence level of 0.95, and on aver- age 4 times better than those predicted by either phylogenetic profiling or gene expression profiling.

  10. Inferring Protein Associations Using Protein Pulldown Assays

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.

    2007-02-01

    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  11. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    2013-01-01

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  12. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  13. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  14. Urine Protein and Urine Protein to Creatinine Ratio

    Science.gov (United States)

    ... products and services. Advertising & Sponsorship: Policy | Opportunities Urine Protein and Urine Protein to Creatinine Ratio Share this page: Was this page helpful? Also known as: 24-Hour Urine Protein; Urine Total Protein; Urine Protein to Creatinine Ratio; ...

  15. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann

    2013-01-01

    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...... facilitates homophilic cell adhesion. Moreover, IGSF9 family proteins have been implicated in the outgrowth and branching of neurites, axon guidance, synapse maturation, self-avoidance, and tiling. However, despite the few published studies on IGSF9 family proteins, reports on the functions of both Turtle...

  16. Tetrabenazine is neuroprotective in Huntington's disease mice

    Directory of Open Access Journals (Sweden)

    Tang Tie-Shan

    2010-04-01

    Full Text Available Abstract Background Huntington's disease (HD is a neurodegenerative disorder caused by a polyglutamine (polyQ expansion in Huntingtin protein (Htt. PolyQ expansion in Httexp causes selective degeneration of striatal medium spiny neurons (MSN in HD patients. A number of previous studies suggested that dopamine signaling plays an important role in HD pathogenesis. A specific inhibitor of vesicular monoamine transporter (VMAT2 tetrabenazine (TBZ has been recently approved by Food and Drug Administration for treatment of HD patients in the USA. TBZ acts by reducing dopaminergic input to the striatum. Results In previous studies we demonstrated that long-term feeding with TBZ (combined with L-Dopa alleviated the motor deficits and reduced the striatal neuronal loss in the yeast artificial chromosome transgenic mouse model of HD (YAC128 mice. To further investigate a potential beneficial effects of TBZ for HD treatment, we here repeated TBZ evaluation in YAC128 mice starting TBZ treatment at 2 months of age ("early" TBZ group and at 6 months of age ("late" TBZ group. In agreement with our previous studies, we found that both "early" and "late" TBZ treatments alleviated motor deficits and reduced striatal cell loss in YAC128 mice. In addition, we have been able to recapitulate and quantify depression-like symptoms in TBZ-treated mice, reminiscent of common side effects observed in HD patients taking TBZ. Conclusions Our results further support therapeutic value of TBZ for treatment of HD but also highlight the need to develop more specific dopamine antagonists which are less prone to side-effects.

  17. Novel Metabolic Abnormalities in the Tricarboxylic Acid Cycle in Peripheral Cells From Huntington’s Disease Patients

    Science.gov (United States)

    Naseri, Nima N.; Bonica, Joseph; Xu, Hui; Park, Larry C.; Arjomand, Jamshid; Chen, Zhengming; Gibson, Gary E.

    2016-01-01

    Metabolic dysfunction is well-documented in Huntington’s disease (HD). However, the link between the mutant huntingtin (mHTT) gene and the pathology is unknown. The tricarboxylic acid (TCA) cycle is the main metabolic pathway for the production of NADH for conversion to ATP via the electron transport chain (ETC). The objective of this study was to test for differences in enzyme activities, mRNAs and protein levels related to the TCA cycle between lymphoblasts from healthy subjects and from patients with HD. The experiments utilize the advantages of lymphoblasts to reveal new insights about HD. The large quantity of homogeneous cell populations permits multiple dynamic measures to be made on exactly comparable tissues. The activities of nine enzymes related to the TCA cycle and the expression of twenty-nine mRNAs encoding for these enzymes and enzyme complexes were measured. Cells were studied under baseline conditions and during metabolic stress. The results support our recent findings that the activities of the pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase (SDH) are elevated in HD. The data also show a large unexpected depression in MDH activities. Furthermore, message levels for isocitrate dehydrogenase 1 (IDH1) were markedly increased in in HD lymphoblasts and were responsive to treatments. The use of lymphoblasts allowed us to clarify that the reported decrease in aconitase activity in HD autopsy brains is likely due to secondary hypoxic effects. These results demonstrate the mRNA and enzymes of the TCA cycle are critical therapeutic targets that have been understudied in HD. PMID:27611087

  18. Functional convergence of Akt protein with VEGFR-1 in human endothelial progenitor cells exposed to sera from patient with type 2 diabetes mellitus.

    Science.gov (United States)

    Hassanpour, Mehdi; Rezabakhsh, Aysa; Rahbarghazi, Reza; Nourazarian, Alireza; Nouri, Mohammad; Avci, Çığır Biray; Ghaderi, Shahrooz; Alidadyani, Neda; Bagca, Bakiye Goker; Bagheri, Hesam Saghaei

    2017-11-01

    Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p<0.001). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p<0.0001) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p<0.0001). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p<0.05). According to data from bioinformatic datasets, we showed VEGFR-1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Physics of protein motility and motor proteins

    Science.gov (United States)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  20. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  1. Protein and protein hydrolysates in sports nutrition.

    Science.gov (United States)

    van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

    2007-08-01

    With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

  2. Protein Data Bank (PDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Protein Data Bank (PDB) archive is the single worldwide repository of information about the 3D structures of large biological molecules, including proteins and...

  3. Protein electrophoresis - serum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003540.htm Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  4. Urine protein electrophoresis test

    Science.gov (United States)

    Urine protein electrophoresis; UPEP; Multiple myeloma - UPEP; Waldenström macroglobulinemia - UPEP; Amyloidosis - UPEP ... special paper and apply an electric current. The proteins move and form visible bands. These reveal the ...

  5. Protein in diet

    Science.gov (United States)

    ... building blocks of life. Every cell in the human body contains protein. The basic structure of protein is ... into parts called amino acids during digestion. The human body needs a number of amino acids in large ...

  6. Abnormal protein aggregationand neurodegenerativediseases

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Abnormal protein aggregation or amyloid is the major cause ofmany neurodegenerative disorders. The present review focuses on the correlation between sequence and structure features of proteins related to the diseases and abnormal protein aggregation. Recent progress has improved our knowledge on understand-ing the mechanism of amyloid formation. We suggest a nucleation model for ordered protein aggregation, which can also explain pathogenesis mechanisms of these neurodegenerative diseases in vivo.

  7. Of proteins and processing

    NARCIS (Netherlands)

    Salazar Villanea, Sergio

    2017-01-01

    Hydrothermal processing is a common practice during the manufacture of protein-rich feed ingredients, such as rapeseed meal (RSM), and feeds. This processing step can induce physical and chemical changes to the proteins, thereby reducing the digestibility and utilization of crude protein (CP) and

  8. Protein Frustratometer 2

    DEFF Research Database (Denmark)

    Gonzalo Parra, R.; Schafer, Nicholas P.; Radusky, Leandro G.

    2016-01-01

    The protein frustratometer is an energy landscape theory-inspired algorithm that aims at localizing and quantifying the energetic frustration present in protein molecules. Frustration is a useful concept for analyzing proteins' biological behavior. It compares the energy distributions of the nati...

  9. Destabilized bioluminescent proteins

    Science.gov (United States)

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  10. CSF total protein

    Science.gov (United States)

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  11. Destabilized bioluminescent proteins

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Michael S. (Knoxville, TN); Rakesh, Gupta (New Delhi, IN); Gary, Sayler S. (Blaine, TN)

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  12. Protein domain prediction

    NARCIS (Netherlands)

    Ingolfsson, Helgi; Yona, Golan

    2008-01-01

    Domains are considered to be the building blocks of protein structures. A protein can contain a single domain or multiple domains, each one typically associated with a specific function. The combination of domains determines the function of the protein, its subcellular localization and the interacti

  13. Protopia: a protein-protein interaction tool

    Science.gov (United States)

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  14. Protein oxidation and peroxidation

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2016-01-01

    and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function....... Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides...

  15. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  16. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  17. Protein crystallization with paper

    Science.gov (United States)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  18. Protein aggregate myopathies

    Directory of Open Access Journals (Sweden)

    Sharma M

    2005-01-01

    Full Text Available Protein aggregate myopathies (PAM are an emerging group of muscle diseases characterized by structural abnormalities. Protein aggregate myopathies are marked by the aggregation of intrinsic proteins within muscle fibers and fall into four major groups or conditions: (1 desmin-related myopathies (DRM that include desminopathies, a-B crystallinopathies, selenoproteinopathies caused by mutations in the, a-B crystallin and selenoprotein N1 genes, (2 hereditary inclusion body myopathies, several of which have been linked to different chromosomal gene loci, but with as yet unidentified protein product, (3 actinopathies marked by mutations in the sarcomeric ACTA1 gene, and (4 myosinopathy marked by a mutation in the MYH-7 gene. While PAM forms 1 and 2 are probably based on impaired extralysosomal protein degradation, resulting in the accumulation of numerous and diverse proteins (in familial types in addition to respective mutant proteins, PAM forms 3 and 4 may represent anabolic or developmental defects because of preservation of sarcomeres outside of the actin and myosin aggregates and dearth or absence of other proteins in these actin or myosin aggregates, respectively. The pathogenetic principles governing protein aggregation within muscle fibers and subsequent structural sarcomeres are still largely unknown in both the putative catabolic and anabolic forms of PAM. Presence of inclusions and their protein composition in other congenital myopathies such as reducing bodies, cylindrical spirals, tubular aggregates and others await clarification. The hitherto described PAMs were first identified by immunohistochemistry of proteins and subsequently by molecular analysis of their genes.

  19. Protein and vegetarian diets.

    Science.gov (United States)

    Marsh, Kate A; Munn, Elizabeth A; Baines, Surinder K

    2013-08-19

    A vegetarian diet can easily meet human dietary protein requirements as long as energy needs are met and a variety of foods are eaten. Vegetarians should obtain protein from a variety of plant sources, including legumes, soy products, grains, nuts and seeds. Eggs and dairy products also provide protein for those following a lacto-ovo-vegetarian diet. There is no need to consciously combine different plant proteins at each meal as long as a variety of foods are eaten from day to day, because the human body maintains a pool of amino acids which can be used to complement dietary protein. The consumption of plant proteins rather than animal proteins by vegetarians may contribute to their reduced risk of chronic diseases such as diabetes and heart disease.

  20. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  1. Overexpression and nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase in a transgenic mouse model of Huntington's disease.

    Science.gov (United States)

    Senatorov, Vladimir V; Charles, Vinod; Reddy, P H; Tagle, Dan A; Chuang, De-Maw

    2003-03-01

    Huntington's disease is due to an expansion of CAG repeats in the huntingtin gene. Huntingtin interacts with several proteins including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We performed immunohistochemical analysis of GAPDH expression in the brains of transgenic mice carrying the huntingtin gene with 89 CAG repeats. In all wild-type animals examined, GAPDH was evenly distributed among the different cell types throughout the brain. In contrast, the majority of transgenic mice showed GAPDH overexpression, with the most prominent GAPDH changes observed in the caudate putamen, globus pallidus, neocortex, and hippocampal formation. Double staining for NeuN and GFAP revealed that GAPDH overexpression occurred exclusively in neurons. Nissl staining analysis of the neocortex and caudate putamen indicated 24 and 27% of cell loss in transgenic mice, respectively. Subcellular fluorescence analysis revealed a predominant increase in GAPDH immunostaining in the nucleus. Thus, we conclude that mutation of huntingtin is associated with GAPDH overexpression and nuclear translocation in discrete populations of brain neurons.

  2. Packing in protein cores

    Science.gov (United States)

    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

    2017-07-01

    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  3. Toxic proteins in plants.

    Science.gov (United States)

    Dang, Liuyi; Van Damme, Els J M

    2015-09-01

    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed.

  4. PROTEIN - WHICH IS BEST?

    Directory of Open Access Journals (Sweden)

    Michael J. Falvo

    2004-09-01

    Full Text Available Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids, whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function are also reviewed

  5. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  6. Protein: FEA4 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA4 Proteins in gibberellin signaling GID2 F-box protein GID2 Gibberellin-insensitive dwarf protein 2, Prot...ein GIBBERELLIN INSENSITIVE DWARF2 39947 Oryza sativa subsp. japonica Q7XAK4 ...

  7. Protein Electrophoresis/Immunofixation Electrophoresis

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Protein Electrophoresis Immunofixation Electrophoresis Share this page: Was this page helpful? Also known as: Serum Protein Electrophoresis; Protein ELP; SPE; SPEP; Urine Protein Electrophoresis; ...

  8. Highly purified, multi-wall carbon nanotubes induce light-chain 3B expression in human lung cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@kanazawa-med.ac.jp [Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 (Japan); Matsuda, Yoshikazu [Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama 362-0806 (Japan); Usui, Yuki [Research Center for Exotic Nanocarbons, Shinshu University, 4-17-1 Wakasato, Nagano-shi, Nagano 380-8553 (Japan); Haniu, Hisao [Department of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

    2013-10-18

    Highlights: •HTT2800-treated BEAS-2B cells induced LC3B in a time-dependent manner. •HTT2800-treated BEAS-2B cells showed decreased cell proliferation that was both time- and dose-dependent. •Addition of 3-MA, LC3B-II protein and mRNA levels were significantly decreased. •3-MA and E64-d + pepstatin A, but not brefeldin A, provided protection against HTT2800-induced cell death. •These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells. -- Abstract: Bronchial epithelial cells are targets of inhalation and play a critical role in the maintenance of mucosal integrity as mechanical barriers against various particles. Our previous result suggest that vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. Increasing evidence suggests that autophagy may critically influence vital cellular processes such as apoptosis, cell proliferation and inflammation and thereby may play a critical role in pulmonary diseases. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles. In this study, the authors focus on the autophagic responses of HTT2800 exposure. The HTT2800-exposed cells induced LC3B expression and induced cell growth inhibition.

  9. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Amarnath Chtterjee; Ashutosh Kumar; Jeetender Chugh; Sudha Srivastava; Neel S Bhavesh; Ramakrishna V Hosur

    2005-01-01

    In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either wholly or in specific regions. It appears that this disorder may be important for regulatory functions of the proteins, on the one hand, and may help in directing the folding process to reach the compact native state, on the other. Nuclear magnetic resonance (NMR) has over the last two decades emerged as the sole, most powerful technique to help characterize these disordered protein systems. In this review, we first discuss the significance of disorder in proteins and then describe the recent developments in NMR methods for their characterization. A brief description of the results obtained on several disordered proteins is presented at the end.

  10. Mayaro virus proteins.

    Science.gov (United States)

    Mezencio, J M; Rebello, M A

    1993-01-01

    Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 +/- 2.3 nm in diameter. Three structural virus proteins were identified and designated p1, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in which three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein synthesized at 5 hours post-infection in both cell lines studied.

  11. Mayaro virus proteins

    Directory of Open Access Journals (Sweden)

    J. M. S. Mezencio

    1993-06-01

    Full Text Available Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%. The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 ñ 2.3 nm in diameter. Three structural virus proteins were identified and designated pl, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected. Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in wich three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein sinthesized at 5 hours post-infection in both cell lines studied.

  12. Protein Models Comparator

    CERN Document Server

    Widera, Paweł

    2011-01-01

    The process of comparison of computer generated protein structural models is an important element of protein structure prediction. It has many uses including model quality evaluation, selection of the final models from a large set of candidates or optimisation of parameters of energy functions used in template free modelling and refinement. Although many protein comparison methods are available online on numerous web servers, their ability to handle a large scale model comparison is often very limited. Most of the servers offer only a single pairwise structural comparison, and they usually do not provide a model-specific comparison with a fixed alignment between the models. To bridge the gap between the protein and model structure comparison we have developed the Protein Models Comparator (pm-cmp). To be able to deliver the scalability on demand and handle large comparison experiments the pm-cmp was implemented "in the cloud". Protein Models Comparator is a scalable web application for a fast distributed comp...

  13. Supramolecular Chemistry Targeting Proteins.

    Science.gov (United States)

    van Dun, Sam; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

    2017-09-28

    The specific recognition of protein surface elements is a fundamental challenge in the life sciences. New developments in this field will form the basis of advanced therapeutic approaches and lead to applications such as sensors, affinity tags, immobilization techniques, and protein-based materials. Synthetic supramolecular molecules and materials are creating new opportunities for protein recognition that are orthogonal to classical small molecule and protein-based approaches. As outlined here, their unique molecular features enable the recognition of amino acids, peptides, and even whole protein surfaces, which can be applied to the modulation and assembly of proteins. We believe that structural insights into these processes are of great value for the further development of this field and have therefore focused this Perspective on contributions that provide such structural data.

  14. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  15. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  16. Trehalose reverses cell malfunction in fibroblasts from normal and Huntington's disease patients caused by proteosome inhibition.

    Directory of Open Access Journals (Sweden)

    Maria Angeles Fernandez-Estevez

    Full Text Available Huntington's disease (HD is a neurodegenerative disorder characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and is caused by polyglutamine expansion in the huntingtin protein. Mutant huntingtin protein and its fragments are resistant to protein degradation and produce a blockade of the ubiquitin proteasome system (UPS. In HD models, the proteasome inhibitor epoxomicin aggravates protein accumulation and the inductor of autophagy, trehalose, diminishes it. We have investigated the effects of epoxomicin and trehalose in skin fibroblasts of control and HD patients. Untreated HD fibroblasts have increased the levels of ubiquitinized proteins and higher levels of reactive oxygen species (ROS, huntingtin and the autophagy marker LAMP2A. Baseline replication rates were higher in HD than in controls fibroblasts but that was reverted after 12 passages. Epoxomicin increases the activated caspase-3, HSP70, huntingtin, ubiquitinated proteins and ROS levels in both HD and controls. Treatment with trehalose counteracts the increase in ROS, ubiquitinated proteins, huntingtin and activated caspase-3 levels induced by epoxomicin, and also increases the LC3 levels more in HD fibroblast than controls. These results suggest that trehalose could revert protein processing abnormalities in patients with Huntington's Disease.

  17. Protein-protein interactions as drug targets.

    Science.gov (United States)

    Skwarczynska, Malgorzata; Ottmann, Christian

    2015-01-01

    Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3.

  18. Acanthamoeba castellanii STAT Protein

    OpenAIRE

    Anna Kicinska; Jacek Leluk; Wieslawa Jarmuszkiewicz

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyos...

  19. Proteins: Form and function

    OpenAIRE

    Roy D Sleator

    2012-01-01

    An overwhelming array of structural variants has evolved from a comparatively small number of protein structural domains; which has in turn facilitated an expanse of functional derivatives. Herein, I review the primary mechanisms which have contributed to the vastness of our existing, and expanding, protein repertoires. Protein function prediction strategies, both sequence and structure based, are also discussed and their associated strengths and weaknesses assessed.

  20. Pressure cryocooling protein crystals

    Science.gov (United States)

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  1. Monkey hybrid stem cells develop cellular features of Huntington's disease

    Directory of Open Access Journals (Sweden)

    Lorthongpanich Chanchao

    2010-02-01

    Full Text Available Abstract Background Pluripotent stem cells that are capable of differentiating into different cell types and develop robust hallmark cellular features are useful tools for clarifying the impact of developmental events on neurodegenerative diseases such as Huntington's disease. Additionally, a Huntington's cell model that develops robust pathological features of Huntington's disease would be valuable for drug discovery research. Results To test this hypothesis, a pluripotent Huntington's disease monkey hybrid cell line (TrES1 was established from a tetraploid Huntington's disease monkey blastocyst generated by the fusion of transgenic Huntington's monkey skin fibroblast and a wild-type non-transgenic monkey oocyte. The TrES1 developed key Huntington's disease cellular pathological features that paralleled neural development. It expressed mutant huntingtin and stem cell markers, was capable of differentiating to neural cells, and developed teratoma in severely compromised immune deficient (SCID mice. Interestingly, the expression of mutant htt, the accumulation of oligomeric mutant htt and the formation of intranuclear inclusions paralleled neural development in vitro , and even mutant htt was ubiquitously expressed. This suggests the development of Huntington's disease cellular features is influenced by neural developmental events. Conclusions Huntington's disease cellular features is influenced by neural developmental events. These results are the first to demonstrate that a pluripotent stem cell line is able to mimic Huntington's disease progression that parallels neural development, which could be a useful cell model for investigating the developmental impact on Huntington's disease pathogenesis.

  2. A broad phenotypic screen identifies novel phenotypes driven by a single mutant allele in Huntington's disease CAG knock-in mice.

    Directory of Open Access Journals (Sweden)

    Sabine M Hölter

    Full Text Available Huntington's disease (HD is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.

  3. Association of age at onset in Huntington disease with functional promoter variations in NPY and NPY2R.

    Science.gov (United States)

    Kloster, Eugen; Saft, Carsten; Akkad, Denis A; Epplen, Jörg T; Arning, Larissa

    2014-02-01

    Huntington disease (HD) is caused by the expansion of a CAG repeat within exon 1 of the HTT gene. Although the variation in age at onset (AO) is partly explained by the lengths of the expanded repeats, the unexplained variation is highly heritable, emphasizing the role of the so-called genetic background on disease expression. Neuropeptide Y (NPY) has been implicated in the modulation of neuroprotection, neurogenesis, and neuroinflammation. Therefore, the aim of the present study was to analyze different single nucleotide polymorphisms (SNPs) in order to test the possibility that genetic variation in NPY or three of its receptor genes (NPY1R, NPY2R, and NPY5R) may explain some of the variation in AO of HD motor manifestations, in a comprehensive cohort of 487 German HD patients. We found modest association of the AO with two NPY promoter variations and a highly significant association with a NPY2R promoter SNP (rs2234759; p = 0.0004). Investigating the functional impact of rs2234759 by luciferase assays revealed that the high-expression NPY2R genotypes were associated with later AO in HD. Additionally, treatment of PC12 cells expressing mutant huntingtin (htt) exon 1 with NPY and the NPY2R agonist NPY(3-36) has a protective effect against mutant htt-induced cell death. Thus, NPY might act through Y2 receptors to slow down the course of HD, and hence, this peptide could be of interest as a possible therapeutic agent.

  4. A broad phenotypic screen identifies novel phenotypes driven by a single mutant allele in Huntington's disease CAG knock-in mice.

    Science.gov (United States)

    Hölter, Sabine M; Stromberg, Mary; Kovalenko, Marina; Garrett, Lillian; Glasl, Lisa; Lopez, Edith; Guide, Jolene; Götz, Alexander; Hans, Wolfgang; Becker, Lore; Rathkolb, Birgit; Rozman, Jan; Schrewed, Anja; Klingenspor, Martin; Klopstock, Thomas; Schulz, Holger; Wolf, Eckhard; Wursta, Wolfgang; Gillis, Tammy; Wakimoto, Hiroko; Seidman, Jonathan; MacDonald, Marcy E; Cotman, Susan; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Lee, Jong-Min; Wheeler, Vanessa C

    2013-01-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.

  5. Huntington disease: a single-gene degenerative disorder of the striatum.

    Science.gov (United States)

    Nopoulos, Peggy C

    2016-03-01

    Huntington disease (HD) is an autosomal dominant, neurodegenerative disorder with a primary etiology of striatal pathology. The Huntingtin gene (HTT) has a unique feature of a DNA trinucleotide (triplet) repeat, with repeat length ranging from 10 to 35 in the normal population. Repeat lengths between 36 and 39 cause HD at reduced penetrance (some will get the disease, others won't) and when expanded to 40 or more repeats (mHTT), causes HD at full penetrance (every person with this length or beyond will definitely develop the disease). The symptoms of HD may be motor, cognitive, and psychiatric, and are consistent with the pathophysiology of frontostriatal circuitry malfunction. Expressed ubiquitously and throughout the entire life cycle (development through adulthood), mHTT causes initial dysfunction and eventual death of a specific cell population within the striatum. Although all areas of the brain are eventually affected, the primary pathology of the disease is regionally specific. As a single-gene disorder, HD has the distinction of having the potential of treatment that is aimed directly at the known pathogenic mechanism by gene silencing, providing hope for neuroprotection and ultimately, prevention.

  6. PIC: Protein Interactions Calculator.

    Science.gov (United States)

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside.

  7. Dietary proteins and angiogenesis.

    Science.gov (United States)

    Medina, Miguel Ángel; Quesada, Ana R

    2014-01-17

    Both defective and persistent angiogenesis are linked to pathological situations in the adult. Compounds able to modulate angiogenesis have a potential value for the treatment of such pathologies. Several small molecules present in the diet have been shown to have modulatory effects on angiogenesis. This review presents the current state of knowledge on the potential modulatory roles of dietary proteins on angiogenesis. There is currently limited available information on the topic. Milk contains at least three proteins for which modulatory effects on angiogenesis have been previously demonstrated. On the other hand, there is some scarce information on the potential of dietary lectins, edible plant proteins and high protein diets to modulate angiogenesis.

  8. [Alternative scaffold proteins].

    Science.gov (United States)

    Petrovskaia, L E; Shingarova, L N; Dolgikh, D A; Kirpichnikov, M P

    2011-01-01

    Review is devoted to the challenging direction in modem molecular biology and bioengineering - the properties of alternative scaffold proteins (ASP) and methods for obtaining ASP binding molecules. ASP molecules incorporate conservative protein core and hypervariable regions, providing for the binding function. Structural classification of ASP includes several types which differ also in their molecular targets and potential applications. Construction of artificial binding proteins on the ASP basis implies a combinatorial library design with subsequent selection of specific binders with the use of phage display or the modem cell-free systems. Alternative binding proteins on non-immunoglobulin scaffolds find broad applications in different fields ofbiotechnology and molecular medicine.

  9. Acanthamoeba castellanii STAT protein.

    Directory of Open Access Journals (Sweden)

    Anna Kicinska

    Full Text Available STAT (signal transducers and activators of transcription proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil, a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  10. Acanthamoeba castellanii STAT protein.

    Science.gov (United States)

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  11. Simulations of Protein Folding

    CERN Document Server

    Cahill, M; Cahill, K E; Cahill, Michael; Fleharty, Mark; Cahill, Kevin

    2000-01-01

    We have developed a simple, phenomenological, Monte-Carlo code that predicts the three-dimensional structure of globular proteins from the DNA sequences that define them. We have applied this code to two small proteins, the villin headpiece (1VII) and cole1 rop (1ROP). Our code folded the 36-residue villin headpiece to a mean rms distance of less than 5 A from its native structure as revealed by NMR; it folded a 56-residue fragment of the protein cole1 rop to within 11 A of its native structure. The denatured starting configurations of these two proteins were, respectively, 29 A and 55 A distant from their native structures.

  12. Moonlighting proteins in cancer.

    Science.gov (United States)

    Min, Kyung-Won; Lee, Seong-Ho; Baek, Seung Joon

    2016-01-01

    Since the 1980s, growing evidence suggested that the cellular localization of proteins determined their activity and biological functions. In a classical view, a protein is characterized by the single cellular compartment where it primarily resides and functions. It is now believed that when proteins appear in different subcellular locations, the cells surpass the expected activity of proteins given the same genomic information to fulfill complex biological behavior. Many proteins are recognized for having the potential to exist in multiple locations in cells. Dysregulation of translocation may cause cancer or contribute to poorer cancer prognosis. Thus, quantitative and comprehensive assessment of dynamic proteins and associated protein movements could be a promising indicator in determining cancer prognosis and efficiency of cancer treatment and therapy. This review will summarize these so-called moonlighting proteins, in terms of a coupled intracellular cancer signaling pathway. Determination of the detailed biological intracellular and extracellular transit and regulatory activity of moonlighting proteins permits a better understanding of cancer and identification of potential means of molecular intervention.

  13. Self assembling proteins

    Science.gov (United States)

    Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

    2004-06-29

    Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

  14. Characterization of Metal Proteins

    Science.gov (United States)

    Unno, Masaki; Ikeda-Saito, Masao

    Some metals are essential for life. Most of these metals are associated with biological macromolecule like DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and more often with proteins: metals bind or interact with them. A number of protein molecules intrinsically contain metals in their structure. Some of these proteins catalyze unique chemical reactions and perform specific physiological functions. In this chapter, we will shed light on the several metalcontaining proteins, termed metalloproteins, and other proteins interacting metals. We will also introduce several key techniques which have been used to characterize these proteins. Characterizing these proteins and to understand the relationships between their structures and functions shall continue to be one of the major avenues to solve the mysteries of life. At first, we introduce what are the protein structures and how these proteins interact with metals. In the next section, we discuss the physiological roles of some representative metals. Next, we show two examples of special metal cofactors those help the biological macromolecules to carry out their functions. Then we describe some functions of metalloproteins. Finally, we introduce some physical methods to characterize metalloproteins.

  15. Ultrafiltration of pegylated proteins

    Science.gov (United States)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  16. Protein: FBB5 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available ependent protein kinase activator A PKR-associated protein X, PKR-associating protein X, Protein activator o...f the interferon-induced protein kinase, Protein kinase, interferon-inducible double stranded RNA-dependent activator 9606 Homo sapiens O75569 8575 2DIX 8575 O75569 ...

  17. Protein Attachment on Nanodiamonds.

    Science.gov (United States)

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  18. Poxviral Ankyrin Proteins

    Directory of Open Access Journals (Sweden)

    Michael H. Herbert

    2015-02-01

    Full Text Available Multiple repeats of the ankyrin motif (ANK are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

  19. Engineered Protein Polymers

    Science.gov (United States)

    2010-05-31

    of each pure polymer, we plan to combine the various polymer solutions in different ratios to tune the composition and physico-chemical properties...protein materials as vehicles for storage and delivery of small molecules. Each protein polymer under concentrations for particle formation ( vida

  20. MODELS OF PROTEIN FOLDING

    Directory of Open Access Journals (Sweden)

    Unnati Ahluwalia

    2012-12-01

    Full Text Available In an attempt to explore the understanding of protein folding mechanism, various models have been proposed in the literature. Advances in recent experimental and computational techniques rationalized our understanding on some of the fundamental features of the protein folding pathways. The goal of this review is to revisit the various models and outline the essential aspects of the folding reaction.

  1. Poxviral Ankyrin Proteins

    Science.gov (United States)

    Herbert, Michael H.; Squire, Christopher J.; Mercer, Andrew A

    2015-01-01

    Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range. PMID:25690795

  2. Advances in Protein Precipitation

    NARCIS (Netherlands)

    Golubovic, M.

    2009-01-01

    Proteins are biological macromolecules, which are among the key components of all living organisms. Proteins are nowadays present in all fields of biotech industry, such as food and feed, synthetic and pharmaceutical industry. They are isolated from their natural sources or produced in different cel

  3. Brushes and proteins

    NARCIS (Netherlands)

    Bosker, W.T.E.

    2011-01-01

      Brushes and Proteins   Wouter T. E. Bosker         Protein adsorption at solid surfaces can be prevented by applying a polymer brush at the surface. A polymer brush consists of polymer chains end-grafted to the surface at such a grafting density that th

  4. Early life stress and serotonin transporter gene variation interact to affect the transcription of the glucocorticoid and mineralocorticoid receptors, and the co-chaperone FKBP5, in the adult rat brain.

    Directory of Open Access Journals (Sweden)

    Rick H. A. Van der Doelen

    2014-10-01

    Full Text Available The short allelic variant of the serotonin transporter (5-HTT promoter-linked polymorphic region (5-HTTLPR has been associated with the etiology of major depression by interaction with early life stress (ELS. A frequently observed endophenotype in depression is the abnormal regulation of levels of stress hormones such as glucocorticoids. It is hypothesized that altered central glucocorticoid influence on stress-related behavior and memory processes could underlie the depressogenic interaction of 5-HTTLPR and ELS. One possible mechanism could be the altered expression of the genes encoding the glucocorticoid and mineralocorticoid receptor (GR, MR and their inhibitory regulator FK506-binding protein 51 (FKBP5 in stress-related forebrain areas. To test this notion, we exposed heterozygous (5-HTT+/- and homozygous (5-HTT-/- serotonin transporter knockout rats and their wildtype littermates (5-HTT+/+ to daily 3 h maternal separations from postnatal day 2 to 14. In the medial prefrontal cortex (mPFC and hippocampus of the adult male offspring, we found that GR, MR and FKBP5 mRNA levels were affected by ELS x 5-HTT genotype interaction. Specifically, 5-HTT+/+ rats exposed to ELS showed decreased GR and FKBP5 mRNA in the dorsal and ventral mPFC, respectively. In contrast, 5-HTT+/- rats showed increased MR mRNA levels in the hippocampus and 5-HTT-/- rats showed increased FKBP5 mRNA in the ventral mPFC after ELS exposure. These findings indicate that 5-HTT genotype determines the specific adaptation of GR, MR and FKBP5 expression in response to early life adversity. Therefore, altered extra-hypothalamic glucocorticoid signaling should be considered to play a role in the depressogenic interaction of ELS and 5-HTTLPR.

  5. Manipulating and Visualizing Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates

  6. Sensitizing properties of proteins

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott

    2014-01-01

    The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

  7. Unconventional protein secretion.

    Science.gov (United States)

    Ding, Yu; Wang, Juan; Wang, Junqi; Stierhof, York-Dieter; Robinson, David G; Jiang, Liwen

    2012-10-01

    It is generally believed that protein secretion or exocytosis is achieved via a conventional ER (endoplasmic reticulum)-Golgi-TGN (trans-Golgi network)-PM (plasma membrane) pathway in the plant endomembrane system. However, such signal peptide (SP)-dependent protein secretion cannot explain the increasing number of SP-lacking proteins which are found outside of the PM in plant cells. The process by which such leaderless secretory proteins (LSPs) gain access to the cell exterior is termed unconventional protein secretion (UPS) and has been well-studied in animal and yeast cells, but largely ignored by the plant community. Here, we review the evidence for UPS in plants especially in regard to the recently discovered EXPO (exocyst-positive-organelle).

  8. Protein Unfolding and Alzheimer's

    Science.gov (United States)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  9. Transdermal delivery of proteins.

    Science.gov (United States)

    Kalluri, Haripriya; Banga, Ajay K

    2011-03-01

    Transdermal delivery of peptides and proteins avoids the disadvantages associated with the invasive parenteral route of administration and other alternative routes such as the pulmonary and nasal routes. Since proteins have a large size and are hydrophilic in nature, they cannot permeate passively across the skin due to the stratum corneum which allows the transport of only small lipophilic drug molecules. Enhancement techniques such as chemical enhancers, iontophoresis, microneedles, electroporation, sonophoresis, thermal ablation, laser ablation, radiofrequency ablation and noninvasive jet injectors aid in the delivery of proteins by overcoming the skin barrier in different ways. In this review, these enhancement techniques that can enable the transdermal delivery of proteins are discussed, including a discussion of mechanisms, sterility requirements, and commercial development of products. Combination of enhancement techniques may result in a synergistic effect allowing increased protein delivery and these are also discussed.

  10. Convergent genetic modulation of the endocrine stress response involves polymorphic variations of 5-HTT, COMT and MAOA

    NARCIS (Netherlands)

    Jabbi, M.; Korf, J.; Kema, I. P.; Hartman, C.; van der Pompe, G.; Minderaa, R. B.; Ormel, J.; den Boer, J. A.

    Highly prevalent stress-related disorders such as major depression ( MD) are characterised by a dysregulation of the neuroendocrine system. Although heritability for these disorders is high, the role of genes in the underlying pathophysiology is poorly understood. Here, we show that polymorphic

  11. Convergent genetic modulation of the endocrine stress response involves polymorphic variations of 5-HTT, COMT and MAOA

    NARCIS (Netherlands)

    Jabbi, M.; Korf, J.; Kema, I. P.; Hartman, C.; van der Pompe, G.; Minderaa, R. B.; Ormel, J.; den Boer, J. A.

    2007-01-01

    Highly prevalent stress-related disorders such as major depression ( MD) are characterised by a dysregulation of the neuroendocrine system. Although heritability for these disorders is high, the role of genes in the underlying pathophysiology is poorly understood. Here, we show that polymorphic vari

  12. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are separate

  13. New Compound Classes: Protein-Protein Interactions.

    Science.gov (United States)

    Ottmann, C

    2016-01-01

    "Protein-protein interactions (PPIs) are one of the most promising new targets in drug discovery. With estimates between 300,000 and 650,000 in human physiology, targeted modulation of PPIs would tremendously extend the "druggable" genome. In fact, in every disease a wealth of potentially addressable PPIs can be found making pharmacological intervention based on PPI modulators in principle a generally applicable technology. An impressing number of success stories in small-molecule PPI inhibition and natural-product PPI stabilization increasingly encourage academia and industry to invest in PPI modulation. In this chapter examples of both inhibition as well as stabilization of PPIs are reviewed including some of the technologies which has been used for their identification."

  14. Anchored design of protein-protein interfaces.

    Directory of Open Access Journals (Sweden)

    Steven M Lewis

    Full Text Available BACKGROUND: Few existing protein-protein interface design methods allow for extensive backbone rearrangements during the design process. There is also a dichotomy between redesign methods, which take advantage of the native interface, and de novo methods, which produce novel binders. METHODOLOGY: Here, we propose a new method for designing novel protein reagents that combines advantages of redesign and de novo methods and allows for extensive backbone motion. This method requires a bound structure of a target and one of its natural binding partners. A key interaction in this interface, the anchor, is computationally grafted out of the partner and into a surface loop on the design scaffold. The design scaffold's surface is then redesigned with backbone flexibility to create a new binding partner for the target. Careful choice of a scaffold will bring experimentally desirable characteristics into the new complex. The use of an anchor both expedites the design process and ensures that binding proceeds against a known location on the target. The use of surface loops on the scaffold allows for flexible-backbone redesign to properly search conformational space. CONCLUSIONS AND SIGNIFICANCE: This protocol was implemented within the Rosetta3 software suite. To demonstrate and evaluate this protocol, we have developed a benchmarking set of structures from the PDB with loop-mediated interfaces. This protocol can recover the correct loop-mediated interface in 15 out of 16 tested structures, using only a single residue as an anchor.

  15. Protein Binding Pocket Dynamics.

    Science.gov (United States)

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  16. PSC: protein surface classification.

    Science.gov (United States)

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-07-01

    We recently proposed to classify proteins by their functional surfaces. Using the structural attributes of functional surfaces, we inferred the pairwise relationships of proteins and constructed an expandable database of protein surface classification (PSC). As the functional surface(s) of a protein is the local region where the protein performs its function, our classification may reflect the functional relationships among proteins. Currently, PSC contains a library of 1974 surface types that include 25,857 functional surfaces identified from 24,170 bound structures. The search tool in PSC empowers users to explore related surfaces that share similar local structures and core functions. Each functional surface is characterized by structural attributes, which are geometric, physicochemical or evolutionary features. The attributes have been normalized as descriptors and integrated to produce a profile for each functional surface in PSC. In addition, binding ligands are recorded for comparisons among homologs. PSC allows users to exploit related binding surfaces to reveal the changes in functionally important residues on homologs that have led to functional divergence during evolution. The substitutions at the key residues of a spatial pattern may determine the functional evolution of a protein. In PSC (http://pocket.uchicago.edu/psc/), a pool of changes in residues on similar functional surfaces is provided.

  17. Protein oxidation in aquatic foods

    DEFF Research Database (Denmark)

    Baron, Caroline P.

    2014-01-01

    The chapter discusses general considerations about protein oxidation and reviews the mechanisms involved in protein oxidation and consequences of protein oxidation on fish proteins. It presents two case studies, the first deals with protein and lipid oxidation in frozen rainbow trout......, and the second with oxidation in salted herring. The mechanisms responsible for initiation of protein oxidation are unclear, but it is generally accepted that free radical species initiating lipid oxidation can also initiate protein oxidation. The chapter focuses on interaction between protein and lipid...... oxidation. The protein carbonyl group measurement is the widely used method for estimating protein oxidation in foods and has been used in fish muscle. The chapter also talks about the impact of protein oxidation on protein functionality, fish muscle texture, and food nutritional value. Protein oxidation...

  18. Bacterial Ice Crystal Controlling Proteins

    Directory of Open Access Journals (Sweden)

    Janet S. H. Lorv

    2014-01-01

    Full Text Available Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

  19. Piezoelectric allostery of protein

    Science.gov (United States)

    Ohnuki, Jun; Sato, Takato; Takano, Mitsunori

    2016-07-01

    Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins.

  20. Alpha Shapes and Proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Sterner, Henrik; Sterner, Peter

    2009-01-01

    We provide a unified description of (weighted) alpha shapes, beta shapes and the corresponding simplicialcomplexes. We discuss their applicability to various protein-related problems. We also discuss filtrations of alpha shapes and touch upon related persistence issues.We claim that the full...... potential of alpha-shapes and related geometrical constructs in protein-related problems yet remains to be realized and verified. We suggest parallel algorithms for (weighted) alpha shapes, and we argue that future use of filtrations and kinetic variants for larger proteins will need such implementation....

  1. Sound of proteins

    DEFF Research Database (Denmark)

    2007-01-01

    In my group we work with Molecular Dynamics to model several different proteins and protein systems. We submit our modelled molecules to changes in temperature, changes in solvent composition and even external pulling forces. To analyze our simulation results we have so far used visual inspection...... and statistical analysis of the resulting molecular trajectories (as everybody else!). However, recently I started assigning a particular sound frequency to each amino acid in the protein, and by setting the amplitude of each frequency according to the movement amplitude we can "hear" whenever two aminoacids...

  2. Protein oxidation and ageing

    DEFF Research Database (Denmark)

    Linton, S; Davies, Michael Jonathan; Dean, R T

    2001-01-01

    of redox-active metal ions that could catalyse oxidant formation. As a result of this decrease in antioxidant defences, and increased rate of ROS formation, it is possible that the impact of ROS increases with age. ROS are known to oxidise biological macromolecules, with proteins an important target....... If the argument that the impact of ROS increases with age is true, then proteins would be expected to accumulate oxidised materials with age, and the rate of such accumulation should increase with time, reflecting impaired inefficiency of homeostasis. Here we review the evidence for the accumulation of oxidised......, or modified, extra- and intra-cellular proteins in vivo....

  3. Protein crystallography prescreen kit

    Science.gov (United States)

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  4. Protein: MPA6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available in 30 kDa adipocyte complement-related protein, Adipocyte complement-related 30 kDa protein, Adipocyte, C1q ...and collagen domain-containing protein, Adipose most abundant gene transcript 1 protein, Gelatin-binding protein 9606 Homo sapiens Q15848 9370 9370 Q15848 18054335, 19646806 ...

  5. Protein: FEB6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEB6 Photoresponse regulatory proteins HD1 SE1 Zinc finger protein HD1 Protein CONSTANS-like, Prot...ein HEADING DATE 1, Protein PHOTOPERIOD SENSITIVITY 1 39947 Oryza sativa subsp. japonica 4340746 Q9FDX8 21952207, 19246394 #shimamoto ...

  6. New approach for predicting protein-protein interactions

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Protein-protein interactions (PPIs) are of vital importance for virtually all processes of a living cell. The study of these associations of protein molecules could improve people's understanding of diseases and provide basis for therapeutic approaches.

  7. Analysis of correlations between protein complex and protein-protein interaction and mRNA expression

    Institute of Scientific and Technical Information of China (English)

    CAI Lun; XUE Hong; LU Hongchao; ZHAO Yi; ZHU Xiaopeng; BU Dongbo; LING Lunjiang; CHEN Runsheng

    2003-01-01

    Protein-protein interaction is a physical interaction of two proteins in living cells. In budding yeast Saccharomyces cerevisiae, large-scale protein-protein interaction data have been obtained through high-throughput yeast two-hybrid systems (Y2H) and protein complex purification techniques based on mass-spectrometry. Here, we collect 11855 interactions between total 2617 proteins. Through seriate genome-wide mRNA expression data, similarity between two genes could be measured. Protein complex data can also be obtained publicly and can be translated to pair relationship that any two proteins can only exist in the same complex or not. Analysis of protein complex data, protein-protein interaction data and mRNA expression data can elucidate correlations between them. The results show that proteins that have interactions or similar expression patterns have a higher possibility to be in the same protein complex than randomized selected proteins, and proteins which have interactions and similar expression patterns are even more possible to exist in the same protein complex. The work indicates that comprehensive integration and analysis of public large-scale bioinformatical data, such as protein complex data, protein-protein interaction data and mRNA expression data, may help to uncover their relationships and common biological information underlying these data. The strategies described here may help to integrate and analyze other functional genomic and proteomic data, such as gene expression profiling, protein-localization mapping and large-scale phenotypic data, both in yeast and in other organisms.

  8. Protein Model Database

    Energy Technology Data Exchange (ETDEWEB)

    Fidelis, K; Adzhubej, A; Kryshtafovych, A; Daniluk, P

    2005-02-23

    The phenomenal success of the genome sequencing projects reveals the power of completeness in revolutionizing biological science. Currently it is possible to sequence entire organisms at a time, allowing for a systemic rather than fractional view of their organization and the various genome-encoded functions. There is an international plan to move towards a similar goal in the area of protein structure. This will not be achieved by experiment alone, but rather by a combination of efforts in crystallography, NMR spectroscopy, and computational modeling. Only a small fraction of structures are expected to be identified experimentally, the remainder to be modeled. Presently there is no organized infrastructure to critically evaluate and present these data to the biological community. The goal of the Protein Model Database project is to create such infrastructure, including (1) public database of theoretically derived protein structures; (2) reliable annotation of protein model quality, (3) novel structure analysis tools, and (4) access to the highest quality modeling techniques available.

  9. Protein urine test

    Science.gov (United States)

    ... Urine albumin; Proteinuria; Albuminuria Images White nail syndrome Protein urine test References Gerber GS, Brendler CB. Evaluation of the urologic patient: history, physical examination, and urinalysis. In: Wein AJ, Kavoussi ...

  10. MicroProteins

    DEFF Research Database (Denmark)

    Eguen, Teinai Ebimienere; Straub, Daniel; Graeff, Moritz;

    2015-01-01

    MicroProteins (miPs) are short, usually single-domain proteins that, in analogy to miRNAs, heterodimerize with their targets and exert a dominant-negative effect. Recent bioinformatic attempts to identify miPs have resulted in a list of potential miPs, many of which lack the defining...... characteristics of a miP. In this opinion article, we clearly state the characteristics of a miP as evidenced by known proteins that fit the definition; we explain why modulatory proteins misrepresented as miPs do not qualify as true miPs. We also discuss the evolutionary history of miPs, and how the miP concept...

  11. The Pentapeptide Repeat Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Vetting,M.; Hegde, S.; Fajardo, J.; Fiser, A.; Roderick, S.; Takiff, H.; Blanchard, J.

    2006-01-01

    The Pentapeptide Repeat Protein (PRP) family has over 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S, T,A, V][D, N][L, F]-[S, T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral {beta}-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable size, shape and electrostatic similarity to DNA.

  12. Interactive protein manipulation

    Energy Technology Data Exchange (ETDEWEB)

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  13. Protein Polymers and Amyloids

    DEFF Research Database (Denmark)

    Risør, Michael Wulff

    2014-01-01

    Several human disorders are caused by a common general disease mechanism arising from abnormal folding and aggregation of the underlying protein. These include the prevalent dementias like Alzheimer’s and Parkinson’s, where accumulation of protein fibrillar structures, known as amyloid fibrils......, is a general hallmark. They also include the α1-antitrypsin deficiency, where disease-causing mutations in the serine protease inhibitor, α1-antitrypsin (α1AT), leads to accumulation of the aberrant protein in the liver of these patients. The native metastable structure of α1AT constitutes a molecular trap...... that inhibits its target protease through a large conformational change but mutations compromise this function and cause premature structural collapse into hyperstable polymers. Understanding the conformational disorders at a molecula