WorldWideScience

Sample records for nitrated alpha synuclein

  1. Alpha-Synuclein to the Rescue: Immune Cell Recruitment by Alpha-Synuclein during Gastrointestinal Infection.

    Science.gov (United States)

    Labrie, Viviane; Brundin, Patrik

    2017-09-02

    Intraneuronal accumulation of misfolded alpha-synuclein in the central and peripheral nervous systems is strongly linked to Parkinson disease (PD) and other related synucleinopathies. In rare inherited forms of PD, point mutations or gene multiplications mediate the formation of alpha-synuclein protein aggregates. However, in most PD cases it is presumed that the combined effects of ageing and environmental factors drive the formation of alpha-synuclein aggregates. Despite advances regarding alpha-synuclein pathobiology, the normal functions of this protein and factors that regulate its expression are not well understood. We discuss a recent study reporting that viral infection induces alpha-synuclein expression in neurons of the gastrointestinal tract. Alpha-synuclein levels increased during norovirus infection in the duodenum of children. In an in vitro paradigm, monomeric and oligomeric alpha-synuclein acted as chemoattractants for neutrophils and monocytes, and promoted the maturation of dendritic cells. This suggests that alpha-synuclein facilitates immune responses to infection. We explore the possibility that intestinal infections, and associated inflammation, place individuals at increased risk of PD by increasing alpha-synuclein levels and promoting the formation of alpha-synuclein aggregates that propagate in a prion-like fashion via the vagal nerve to the brainstem. © 2017 S. Karger AG, Basel.

  2. Alpha synuclein in Parkinson's disease

    DEFF Research Database (Denmark)

    Kragh, Christine Lund; Romero-Ramos, Marina; Halliday, Glenda M

    2014-01-01

    The perception of Parkinson’s disease (PD) as a disease centered on dopaminergic striatonigral neurodegeneration has changed fundamentally since 1997 when the first mutation in the SNCA gene (PARK1) encoding a-synuclein was discovered (Polymeropoulos et al. 1997). This discovery formed the basis...... the last decade (Gai et al. 1998; Spillantini and Goedert 2000; Huang et al. 2004; Ubhi et al. 2011). This review will briefly highlight the historical breakthroughs but focus on a-synuclein modifications, human neuropathology, biomarker potential, current animal models and the new concepts emerging after...

  3. Alpha synuclein in Parkinson's disease

    DEFF Research Database (Denmark)

    Kragh, Christine Lund; Romero-Ramos, Marina; Halliday, Glenda M

    2014-01-01

    The perception of Parkinson’s disease (PD) as a disease centered on dopaminergic striatonigral neurodegeneration has changed fundamentally since 1997 when the first mutation in the SNCA gene (PARK1) encoding a-synuclein was discovered (Polymeropoulos et al. 1997). This discovery formed the basis...

  4. Unique copper-induced oligomers mediate alpha-synuclein toxicity.

    Science.gov (United States)

    Wright, Josephine A; Wang, Xiaoyan; Brown, David R

    2009-08-01

    Parkinson's disease and a number of other neurodegenerative diseases have been linked to either genetic mutations in the alpha-synuclein gene or show evidence of aggregates of the alpha-synuclein protein, sometimes in the form of Lewy bodies. There currently is no clear evidence of a distinct neurotoxic species of alpha-synuclein to explain the death of neurons in these diseases. We undertook to assess the toxicity of alpha-synuclein via exogenous application in cell culture. Initially, we showed that only aggregated alpha-synuclein is neurotoxic and requires the presence copper but not iron. Other members of the synuclein family showed no toxicity in any form and inherited point mutations did not alter the effective toxic concentration of alpha-synuclein. Through protein fractionation techniques, we were able to isolate an oligomeric species responsible for the toxicity of alpha-synuclein. This oligomeric species has a unique stellate appearance under EM and again, requires association with copper to induce cell death. The results allow us to suggest that the toxic species of alpha-synuclein in vivo could possibly be these stellate oligomers and not fibrils. Our data provide a link between the recently noted association of copper and alpha-synuclein and a potential role for the combination in causing neurodegeneration.

  5. alpha-synuclein and LRRK2: partners in crime.

    Science.gov (United States)

    Tong, Youren; Shen, Jie

    2009-12-24

    In this issue of Neuron, Lin et al. report that LRRK2 modulates age-related neurodegeneration caused by overexpression of alpha-synuclein in the forebrain of transgenic mice. Overexpression of LRRK2 accelerates the progression of alpha-synuclein-mediated neuropathological changes, whereas deletion of LRRK2 alleviates these alterations. The results reveal an interesting interaction between alpha-synuclein and LRRK2, two gene products linked to dominantly inherited Parkinson's disease.

  6. Alpha-synuclein and Parkinson disease

    Institute of Scientific and Technical Information of China (English)

    Hui Liu; Xiaozhong Wang

    2007-01-01

    OBJECTIVE: To review the recent progresses on the studies of α-synuclein in the pathogenesis of Parkinson disease (PD) and look into the perspective of α-synuclein as a new therapy target.DATA SOURCES: To search the literatures on the progresses of PD studies, especially on the structure, gene expression of α-synuclein and the pathogenesis of PD in Medline from January 1998 to February 2007.Search terms were "Parkinson's disease, α -synuclein" in English.STUDY SELECTION: Initial check the data and choose the original and review articles directly linked to the role of α -synuclein in PD pathogenesis and screening out indirectly discussing articles. Collect the full text and trace the quoting articles and the quoted articles. Only the latest reviews were chosen in Chinese articles.DATA EXTRACTION: There were 424 articles on α-synuclein and its role in the pathogenesis of PD and 43 articles directly related with α -synuclein were chosen among which 12 were reviews.DATA SYNTHESIS: α-synuclein is a kind of soluble protein expressed in pre-synapse in central nervous system encoded by gene in homologous chromosome 4q21. It has physiological function in modulating the stability of membrane and neural plasticity. There is a close relationship between gene mutation in α -synuclein and the pathogenesis of PD. Environmental and genetic factors can induce the misfolding of α-synuclein, and secondary structural change can result in oligomer formation which induces a series of cascade reaction to damage dopaminergic system subsequently. Cell and animal transgenic and non-transgenic models are established recently and the important role of α -synuclein in the pathogenesis both of familial and sporadic PD is confirmed. Studies reveal that inhibiting the aggregation of α -synuclein can prevent its neurotoxicity;gene parkin can intercept the cell death pathway triggered by the aggregation of α -synuclein in cytoplasm.CONCLUSION: Gene mutation of α -synuclein and the

  7. Alpha-synuclein in cutaneous small nerve fibers

    Science.gov (United States)

    Siepmann, Timo; Illigens, Ben Min-Woo; Barlinn, Kristian

    2016-01-01

    Despite progression in the development of pharmacological therapy, treatment of alpha synucleinopathies, such as Parkinson’s disease (PD) and some atypical parkinsonism syndromes, is still challenging. To date, our knowledge of the mechanisms whereby the pathological form of alpha-synuclein causes structural and functional damage to the nervous system is limited and, consequently, there is a lack of specific diagnostic tools to evaluate pathology in these patients and differentiate PD from other neurodegenerative proteinopathies. Recent studies indicated that alpha-synuclein deposition in cutaneous small nerve fibers assessed by skin biopsies might be a valid disease marker of PD and facilitate early differentiation of PD from atypical parkinsonism syndromes. This observation is relevant since early diagnosis may enable timely treatment and improve quality of life. However, challenges include the necessity of standardizing immunohistochemical analysis techniques and the identification of potential distinct patterns of intraneural alpha-synuclein deposition among synucleinopathies. In this perspective, we explore the scientific and clinical opportunities arising from alpha-synuclein assessment using skin biopsies. These include elucidation of the peripheral nervous system pathology of PD and other synucleinopathies, identification of novel targets to study response to neuroprotective treatment, and improvement of clinical management. Furthermore, we discuss future challenges in exploring the diagnostic value of skin biopsy assessment for alpha-synuclein deposition and implementing the technique in clinical practice. PMID:27822045

  8. Prion-like propagation of human brain-derived alpha-synuclein in transgenic mice expressing human wild-type alpha-synuclein.

    Science.gov (United States)

    Bernis, Maria E; Babila, Julius T; Breid, Sara; Wüsten, Katharina Annick; Wüllner, Ullrich; Tamgüney, Gültekin

    2015-11-26

    Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases that are characterized by the intracellular accumulation of alpha-synuclein containing aggregates. Recent increasing evidence suggests that Parkinson's disease and MSA pathology spread throughout the nervous system in a spatiotemporal fashion, possibly by prion-like propagation of alpha-synuclein positive aggregates between synaptically connected areas. Concurrently, intracerebral injection of pathological alpha-synuclein into transgenic mice overexpressing human wild-type alpha-synuclein, or human alpha-synuclein with the familial A53T mutation, or into wild-type mice causes spreading of alpha-synuclein pathology in the CNS. Considering that wild-type mice naturally also express a threonine at codon 53 of alpha-synuclein, it has remained unclear whether human wild-type alpha-synuclein alone, in the absence of endogenously expressed mouse alpha-synuclein, would support a similar propagation of alpha-synuclein pathology in vivo. Here we show that brain extracts from two patients with MSA and two patients with probable incidental Lewy body disease (iLBD) but not phosphate-buffered saline induce prion-like spreading of pathological alpha-synuclein after intrastriatal injection into mice expressing human wild-type alpha-synuclein. Mice were sacrificed at 3, 6, and 9 months post injection and analyzed neuropathologically and biochemically. Mice injected with brain extracts from patients with MSA or probable iLBD both accumulated intraneuronal inclusion bodies, which stained positive for phosphorylated alpha-synuclein and appeared predominantly within the injected brain hemisphere after 6 months. After 9 months these intraneuronal inclusion bodies had spread to the contralateral hemisphere and more rostral and caudal areas. Biochemical analysis showed that brains of mice injected with brain extracts from patients with MSA and probable iLBD contained hyperphosphorylated alpha-synuclein

  9. Peculiarities of copper binding to alpha-synuclein.

    Science.gov (United States)

    Ahmad, Atta; Burns, Colin S; Fink, Anthony L; Uversky, Vladimir N

    2012-01-01

    Heavy metals have been implicated as the causative agents for the pathogenesis of the most prevalent neurodegenerative disease. Various mechanisms have been proposed to explain the toxic effects of metals ranging from metal-induced oxidation of protein to metal-induced changes in the protein conformation. Aggregation of a-synuclein is implicated in Parkinson's disease (PD), and various metals, including copper, constitute a prominent group of alpha-synuclein aggregation enhancers. In this study, we have systematically characterized the a-synuclein-Cu21 binding sites and analyzed the possible role of metal binding in a-synuclein fibrillation using a set of biophysical techniques, such as electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), circular dichroism (CD), and size exclusion chromatography (SEC). Our analyses indicated that a-synuclein possesses at least two binding sites for Cu21. We have been able to locate one of the binding sites in the N-terminal region. Furthermore, based on the EPR studies of model peptides and Beta-synuclein, we concluded that the suspected His residue did not appear to participate in strong Cu21 binding.

  10. Alpha-synuclein in cutaneous small nerve fibers

    Directory of Open Access Journals (Sweden)

    Siepmann T

    2016-10-01

    Full Text Available Timo Siepmann,1 Ben Min-Woo Illigens,2 Kristian Barlinn1 1Department of Neurology, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; 2Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Abstract: Despite progression in the development of pharmacological therapy, treatment of alpha synucleinopathies, such as Parkinson’s disease (PD and some atypical parkinsonism syndromes, is still challenging. To date, our knowledge of the mechanisms whereby the pathological form of alpha-synuclein causes structural and functional damage to the nervous system is limited and, consequently, there is a lack of specific diagnostic tools to evaluate pathology in these patients and differentiate PD from other neurodegenerative proteinopathies. Recent studies indicated that alpha-synuclein deposition in cutaneous small nerve fibers assessed by skin biopsies might be a valid disease marker of PD and facilitate early differentiation of PD from atypical parkinsonism syndromes. This observation is relevant since early diagnosis may enable timely treatment and improve quality of life. However, challenges include the necessity of standardizing immunohistochemical analysis techniques and the identification of potential distinct patterns of intraneural alpha-synuclein deposition among synucleinopathies. In this perspective, we explore the scientific and clinical opportunities arising from alpha-synuclein assessment using skin biopsies. These include elucidation of the peripheral nervous system pathology of PD and other synucleinopathies, identification of novel targets to study response to neuroprotective treatment, and improvement of clinical management. Furthermore, we discuss future challenges in exploring the diagnostic value of skin biopsy assessment for alpha-synuclein deposition and implementing the technique in clinical practice. Keywords: Parkinson’s disease, diagnosis, skin

  11. alpha-Synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or toxicity.

    Science.gov (United States)

    Brandis, Katrina A; Holmes, Isaac F; England, Samantha J; Sharma, Nijee; Kukreja, Lokesh; DebBurman, Shubhik K

    2006-01-01

    Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis.

  12. Alpha-synuclein suppression by targeted small interfering RNA in the primate substantia nigra.

    Directory of Open Access Journals (Sweden)

    Alison L McCormack

    Full Text Available The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left vs. untreated (right hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i the number and phenotype of nigral dopaminergic neurons, and (ii the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

  13. Familial Parkinson mutant alpha-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans.

    Science.gov (United States)

    Kuwahara, Tomoki; Koyama, Akihiko; Gengyo-Ando, Keiko; Masuda, Mayumi; Kowa, Hisatomo; Tsunoda, Makoto; Mitani, Shohei; Iwatsubo, Takeshi

    2006-01-06

    Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.

  14. Alpha-synuclein gene deletion decreases brain palmitate uptake and alters the palmitate metabolism in the absence of alpha-synuclein palmitate binding

    DEFF Research Database (Denmark)

    Golovko, Mikhail Y; Færgeman, Nils J.; Cole, Nelson B;

    2005-01-01

    . To better define a role for alpha-synuclein in brain fatty acid uptake and metabolism, we infused awake, wild-type, or alpha-synuclein gene-ablated mice with [1-(14)C]palmitic acid (16:0) and assessed fatty acid uptake and turnover kinetics in brain phospholipids. Alpha-synuclein deficiency decreased brain......Alpha-synuclein is an abundant protein in the central nervous system that is associated with a number of neurodegenerative disorders, including Parkinson's disease. Its physiological function is poorly understood, although recently it was proposed to function as a fatty acid binding protein...... 16:0 uptake 35% and reduced its targeting to the organic fraction. The incorporation coefficient for 16:0 entering the brain acyl-CoA pool was significantly decreased 36% in alpha-synuclein gene-ablated mice. Because incorporation coefficients alone are not predictive of fatty acid turnover...

  15. Alpha-synuclein is a cellular ferrireductase.

    Directory of Open Access Journals (Sweden)

    Paul Davies

    Full Text Available α-synuclein (αS is a cellular protein mostly known for the association of its aggregated forms with a variety of diseases that include Parkinson's disease and Dementia with Lewy Bodies. While the role of αS in disease is well documented there is currently no agreement on the physiological function of the normal isoform of the protein. Here we provide strong evidence that αS is a cellular ferrireductase, responsible for reducing iron (III to bio available iron (II. The recombinant form of the protein has a V(Max of 2.72 nmols/min/mg and K(m 23 µM. This activity is also evident in lysates from neuronal cell lines overexpressing αS. This activity is dependent on copper bound to αS as a cofactor and NADH as an electron donor. Overexpression of α-synuclein by cells significantly increases the percentage of iron (II in cells. The common disease mutations associated with increased susceptibility to PD show no [corrected] differences in activity or iron (II levels. This discovery may well provide new therapeutic targets for PD and Lewy body dementias.

  16. Alpha-synuclein levels in blood plasma decline with healthy aging.

    Science.gov (United States)

    Koehler, Niklas K U; Stransky, Elke; Meyer, Mirjam; Gaertner, Susanne; Shing, Mona; Schnaidt, Martina; Celej, Maria S; Jovin, Thomas M; Leyhe, Thomas; Laske, Christoph; Batra, Anil; Buchkremer, Gerhard; Fallgatter, Andreas J; Wernet, Dorothee; Richartz-Salzburger, Elke

    2015-01-01

    There is unequivocal evidence that alpha-synuclein plays a pivotal pathophysiological role in neurodegenerative diseases, and in particular in synucleinopathies. These disorders present with a variable extent of cognitive impairment and alpha-synuclein is being explored as a biomarker in CSF, blood serum and plasma. Considering key events of aging that include proteostasis, alpha-synuclein may not only be useful as a marker for differential diagnosis but also for aging per se. To explore this hypothesis, we developed a highly specific ELISA to measure alpha-synuclein. In healthy males plasma alpha-synuclein levels correlated strongly with age, revealing much lower concentrations in older (avg. 58.1 years) compared to younger (avg. 27.6 years) individuals. This difference between the age groups was enhanced after acidification of the plasmas (phealthy aging. Thus, alpha-synuclein may be a novel biomarker of aging, a factor that should be considered when analyzing its presence in biological specimens.

  17. Molecular understanding of copper and iron interaction with alpha-synuclein by fluorescence analysis.

    Science.gov (United States)

    Bharathi; Rao, K S J

    2008-07-01

    Alpha-synuclein aggregation is a hallmark pathological feature in Parkinson's disease (PD). The conversion of alpha-synuclein from a soluble monomer to an insoluble fibril may underlie the neurodegeneration associated with PD. Redox-active metal ions such as iron (Fe) and copper (Cu) are known to enhance alpha-synuclein fibrillogenesis. In the present investigation, we analyzed the binding efficiency of Cu and Fe to alpha-synuclein by fluorescence studies. It is interesting to note that Cu and Fe showed differential binding pattern toward alpha-synuclein (wild type and A30P, A53T, and E46K mutant forms) as revealed by intrinsic tyrosine fluorescence, thioflavin-T fluorescence, 1-anilino-8-naphthalenesulfonate-binding studies, and scatchard plot analysis. The experimental data might prove useful in understanding the hierarchy of metals binding to alpha-synuclein and its role in neurodegeneration.

  18. alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress.

    Science.gov (United States)

    Sharma, Nijee; Brandis, Katrina A; Herrera, Sara K; Johnson, Brandon E; Vaidya, Tulaza; Shrestha, Ruja; Debburman, Shubhik K

    2006-01-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity.

  19. Mutant and wild-type alpha-synuclein interact with mitochondrial cytochrome C oxidase.

    Science.gov (United States)

    Elkon, Hanock; Don, Jermy; Melamed, Eldad; Ziv, Ilan; Shirvan, Anat; Offen, Daniel

    2002-06-01

    Alpha-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in both inherited and sporadic Parkinson's disease (PD). Furthermore, rare mutations of alpha-synuclein cause autosomal-dominant PD. However, it is unknown how alpha-synuclein is involved in the pathogenesis of nigral degeneration in PD. In this study, we examine the protein-protein interactions of wild-type and mutant (A53T) a-synuclein with adult human brain cDNA expression library using the yeast two-hybrid technique. We found that both normal and mutant alpha-synuclein specifically interact with the mitochondrial complex IV enzyme, cytochrome C oxidase (COX). Wild-type and mutant alpha-synuclein genes were further fused with c-Myc tag and translated in rabbit reticulocyte lysate. Using anti-c-Myc antibody, we demonstrated that both wild-type and mutant alpha-synuclein, coimmunoprecipitated with COX. We also showed that potassium cyanide, a selective COX inhibitor, synergistically enhanced the sensitivity of SH-SY5Y neuroblastoma cells to dopamine-induced cell death. In conclusion, we found specific protein-protein interactions of alpha-synuclein, a major LB protein, to COX, a key enzyme of the mithochondrial respiratory system. This interaction suggests that alpha-synuclein aggregation may contribute to enhance the mitochondrial dysfunction, which might be a key factor in the pathogenesis of PD.

  20. Increased CSF alpha-synuclein levels in Alzheimer's disease : Correlation with tau levels

    NARCIS (Netherlands)

    Slaets, Sylvie; Vanmechelen, Eugeen; Le Bastard, Nathalie; Decraemer, Hilde; Vandijck, Manu; Martin, Jean-Jacques; De Deyn, Peter Paul; Engelborghs, Sebastiaan

    2014-01-01

    Background: Given the difficult clinical differential diagnosis between Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), growing interest resulted in research on alpha-synuclein as a potential cerebrospinal fluid biomarker (CSF) for synucleinopathies. Methods: CSF alpha-synuclein-140 co

  1. alpha-Synuclein enhances secretion and toxicity of amyloid beta peptides in PC12 cells

    NARCIS (Netherlands)

    Kazmierczak, Anna; Strosznajder, Joanna B.; Adamczyk, Agata

    2008-01-01

    alpha-Synuclein is the fundamental component of Lewy bodies which occur in the brain of 60% of sporadic and familial Alzheimer's disease patients. Moreover, a proteolytic fragment of alpha-synuclein, the so-called non-amyloid component of Alzheimer's disease amyloid, was found to be an integral part

  2. Exosomes of BV-2 cells induced by alpha-synuclein: important mediator of neurodegeneration in PD.

    Science.gov (United States)

    Chang, Chongwang; Lang, Hongjuan; Geng, Ning; Wang, Jing; Li, Nan; Wang, Xuelian

    2013-08-26

    Parkinson's disease (PD) is a progressive neurodegenerative disease. Alpha-synuclein aggregation, which can activate microglia to enhance its dopaminergic neurotoxicity, plays a central role in the progression of PD. However the mechanism is still unclear. To investigate how alpha-synuclein affects the neuron, exosomes were derived from alpha-synuclein treated mouse microglia cell line BV-2 cells by differential centrifugation and ultracentrifugation. We found that alpha-synuclein can induce an increase of exosomal secretion by microglia. These activated exosomes expressed a high level of MHC class II molecules and membrane TNF-α. In addition, the activated exosomes cause increased apoptosis. Exosomes secreted from activated microglias might be important mediator of alpha-synuclein-induced neurodegeneration in PD.

  3. Testosterone regulates alpha-synuclein mRNA in the avian song system.

    Science.gov (United States)

    Hartman, V N; Miller, M A; Clayton, D F; Liu, W C; Kroodsma, D E; Brenowitz, E A

    2001-04-17

    Alpha-synuclein is a small, highly conserved protein in vertebrates that has been linked to several neurodegenerative diseases. The avian song control system is one of the model systems in which the protein was independently discovered. Alpha-synuclein is dynamically regulated in the song system during song learning, a process in which sex steroids play a central role. We compared alpha-synuclein mRNA expression in the brains of 12 adult male chipping sparrows (Spizella passerina) treated with either testosterone or blank s.c. implants. We saw pronounced upregulation of alpha-synuclein mRNA in, as well as an increase in the volume of, the song control nucleus area X in response to exogenous testosterone. To our knowledge this is the first report of steroid regulation of synuclein gene expression in any model system.

  4. Structure, function and toxicity of alpha-synuclein: the Bermuda triangle in synucleinopathies.

    Science.gov (United States)

    Villar-Piqué, Anna; Lopes da Fonseca, Tomás; Outeiro, Tiago Fleming

    2016-10-01

    Parkinson's disease belongs to a group of currently incurable neurodegenerative disorders characterized by the misfolding and accumulation of alpha-synuclein aggregates that are commonly known as synucleinopathies. Clinically, synucleinopathies are heterogeneous, reflecting the somewhat selective neuronal vulnerability characteristic of each disease. The precise molecular underpinnings of synucleinopathies remain unclear, but the process of aggregation of alpha-synuclein appears as a central event. However, there is still no consensus with respect to the toxic forms of alpha-synuclein, hampering our ability to use the protein as a target for therapeutic intervention. To decipher the molecular bases of synucleinopathies, it is essential to understand the complex triangle formed between the structure, function and toxicity of alpha-synuclein. Recently, important steps have been undertaken to elucidate the role of the protein in both physiological and pathological conditions. Here, we provide an overview of recent findings in the field of alpha-synuclein research, and put forward a new perspective over paradigms that persist in the field. Establishing whether alpha-synuclein has a causative role in all synucleinopathies will enable the identification of targets for the development of novel therapeutic strategies for this devastating group of disorders. Alpha-synuclein is the speculated cornerstone of several neurodegenerative disorders known as Synucleinopathies. Nevertheless, the mechanisms underlying the pathogenic effects of this protein remain unknown. Here, we review the recent findings in the three corners of alpha-synuclein biology - structure, function and toxicity - and discuss the enigmatic roads that have accompanied alpha-synuclein from the beginning. This article is part of a special issue on Parkinson disease.

  5. Alpha-synuclein gene structure,evolution,and protein aggregation

    Institute of Scientific and Technical Information of China (English)

    Lili Xiong; Peng Zhao; Zhiyun Guo; Jianhua Zhang; Diqiang Li; Canquan Mao

    2010-01-01

    α-synuclein,a member of the synuclein family,is predominately expressed in brain tissues,where it is the major component of Lewy bodies,the major hallmark of Parkinson's disease.We analyzed the phylogenetics,gene structure,and effects of different forms of α-synuclein on in vitro protein aggregation.The synuclein phylogenetic tree showed that sequences could be classified into α,β,and γ protein groups.The orthologous gene α-,β-and γ-synuclein showed similar evolutionary distance to the paralogous gene α-,β-and γ-synuclein.Bioinformatics analysis suggests that the amino-acid sequence of human α-synuclein can be divided into three regions: N-terminal amphipathic region(1-60),central hydrophobic non-amyloid beta component segment(61-95),and the C-terminal acidic part(96-140).The mutant site of A30P is at the second exon of α-synuclein,whereas E46K is located at the third exon of α-synuclein.α-synuclein alternative splicing results in four isomers,and five exons,all of which participate in protein coding,comprising 140 amino acids to produce the major α-synuclein in vivo.The threeα-synuclein isoforms are products of alternative splicing,α-synuclein 126,112 and 98.We also review the genetic and cellular factors that affect the aggregation of α-synuclein and compounds that inhibit aggregation.A better understanding of α-synuclein sequences,structure,and function may allow better targeted therapy and diagnosis of α-synuclein in Parkinson's disease and other neurodegenerative diseases.

  6. Drift diffusion model of reward and punishment learning in rare alpha-synuclein gene carriers.

    Science.gov (United States)

    Moustafa, Ahmed A; Kéri, Szabolcs; Polner, Bertalan; White, Corey

    2017-03-20

    To understand the cognitive effects of alpha-synuclein polymorphism, we employed a drift diffusion model (DDM) to analyze reward- and punishment-guided probabilistic learning task data of participants with the rare alpha-synuclein gene duplication and age- and education-matched controls. Overall, the DDM analysis showed that, relative to controls, asymptomatic alpha-synuclein gene duplication carriers had significantly increased learning from negative feedback, while they tended to show impaired learning from positive feedback. No significant differences were found in response caution, response bias, or motor/encoding time. We here discuss the implications of these computational findings to the understanding of the neural mechanism of alpha-synuclein gene duplication.

  7. DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson's disease.

    Science.gov (United States)

    Hong, Zhen; Shi, Min; Chung, Kathryn A; Quinn, Joseph F; Peskind, Elaine R; Galasko, Douglas; Jankovic, Joseph; Zabetian, Cyrus P; Leverenz, James B; Baird, Geoffrey; Montine, Thomas J; Hancock, Aneeka M; Hwang, Hyejin; Pan, Catherine; Bradner, Joshua; Kang, Un J; Jensen, Poul H; Zhang, Jing

    2010-03-01

    Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in Parkinson's disease. Both DJ-1 and alpha-synuclein, two proteins critically involved in Parkinson's disease pathogenesis, have been tested as disease biomarkers in several recent studies with inconsistent results. These have been largely due to variation in the protein species detected by different antibodies, limited numbers of patients in some studies, or inadequate control of several important variables. In this study, the nature of DJ-1 and alpha-synuclein in human cerebrospinal fluid was studied by a combination of western blotting, gel filtration and mass spectrometry. Sensitive and quantitative Luminex assays detecting most, if not all, species of DJ-1 and alpha-synuclein in human cerebrospinal fluid were established. Cerebrospinal fluid concentrations of DJ-1 and alpha-synuclein from 117 patients with Parkinson's disease, 132 healthy individuals and 50 patients with Alzheimer's disease were analysed using newly developed, highly sensitive Luminex technology while controlling for several major confounders. A total of 299 individuals and 389 samples were analysed. The results showed that cerebrospinal fluid DJ-1 and alpha-synuclein levels were dependent on age and influenced by the extent of blood contamination in cerebrospinal fluid. Both DJ-1 and alpha-synuclein levels were decreased in Parkinson's patients versus controls or Alzheimer's patients when blood contamination was controlled for. In the population aged > or = 65 years, when cut-off values of 40 and 0.5 ng/ml were chosen for DJ-1 and alpha-synuclein, respectively, the sensitivity and specificity for patients with Parkinson's disease versus controls were 90 and 70% for DJ-1, and 92 and 58% for alpha-synuclein. A combination of the two markers did not enhance the test performance. There was no association between DJ-1 or alpha-synuclein and the severity of Parkinson's disease. Taken together, this represents

  8. Alpha-synuclein in blood and brain from familial Parkinson disease with SNCA locus triplication.

    Science.gov (United States)

    Miller, D W; Hague, S M; Clarimon, J; Baptista, M; Gwinn-Hardy, K; Cookson, M R; Singleton, A B

    2004-05-25

    The authors recently demonstrated that genetic triplication of the SNCA locus causes Parkinson disease. Here it is shown that SNCA triplication results in a doubling in the amount of alpha-synuclein protein in blood. Examination of brain tissue showed a doubling in the level of SNCA message. However, at the protein level in brain, there was a greater effect on deposition of aggregated forms into insoluble fractions than on net expression of soluble alpha-synuclein.

  9. Alpha-synuclein association with phosphatidylglycerol probed by lipid spin labels.

    Science.gov (United States)

    Ramakrishnan, Muthu; Jensen, Poul H; Marsh, Derek

    2003-11-11

    Alpha-synuclein is a small presynaptic protein, which is linked to the development of Parkinson's disease. Alpha-synuclein partitions between cytosolic and vesicle-bound states, where membrane binding is accompanied by the formation of an amphipathic helix in the N-terminal section of the otherwise unstructured protein. The impact on alpha-synuclein of binding to vesicle-like liposomes has been studied extensively, but far less is known about the impact of alpha-synuclein on the membrane. The interactions of alpha-synuclein with phosphatidylglycerol membranes are studied here by using spin-labeled lipid species and electron spin resonance (ESR) spectroscopy to allow a detailed analysis of the effect on the membrane lipids. Membrane association of alpha-synuclein perturbs the ESR spectra of spin-labeled lipids in bilayers of phosphatidylglycerol but not of phosphatidylcholine. The interaction is inhibited at high ionic strength. The segmental motion is hindered at all positions of spin labeling in the phosphatidylglycerol sn-2 chain, while still preserving the chain flexibility gradient characteristic of fluid phospholipid membranes. Direct motional restriction of the lipid chains, resulting from penetration of the protein into the hydrophobic interior of the membrane, is not observed. Saturation occurs at a protein/lipid ratio corresponding to approximately 36 lipids/protein added. Alpha-synuclein exhibits a selectivity of interaction with different phospholipid spin labels when bound to phosphatidylglycerol membranes in the following order: stearic acid > cardiolipin > phosphatidylcholine > phosphatidylglycerol approximately phosphatidylethanolamine > phosphatidic acid approximately phosphatidylserine > N-acyl phosphatidylethanolamine > diglyceride. Accordingly, membrane-bound alpha-synuclein associates at the interfacial region of the bilayer where it may favor a local concentration of certain phospholipids.

  10. C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging.

    Directory of Open Access Journals (Sweden)

    Tjakko J van Ham

    2008-03-01

    Full Text Available Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha- synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders.

  11. Mutant alpha-synuclein and autophagy in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Kangyong Liu; Chunfeng Liu; Chuancheng Ren; Yaping Yang; Liwei Shen; Xuezhong Li; Fen Wang; Zhenghong Qin

    2011-01-01

    Several studies have demonstrated that overexpression of mutant α-synuclein in PC12 cells is related to occurrence of autophagy.The present study established mutant a-synuclein (A30P)-transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively.Results demonstrated that mutant o-synuclein resulted in cell death via autophagy and involved α-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity.Mutant α-synuclein (A30P) also mediated toxicity of1-methyl-4-phenylpyridinium ion.Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted o-synuclein aggregation and cell death.To further determine the role of autophagy due to mutant a-synuclein, the present study measured expression of microtubule-associated protein light chain 3.Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3,while rapamycin promoted its expression.These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.

  12. Alpha-synuclein in motor neuron disease: an immunohistologic study.

    Science.gov (United States)

    Doherty, M J; Bird, T D; Leverenz, J B

    2004-02-01

    Alpha-synuclein (ASN) has been implicated in neurodegenerative disorders characterized by Lewy body inclusions such as Parkinson's disease and dementia with Lewy bodies. Lewy body-like inclusions have also been observed in spinal neurons of patients with amyotrophic lateral sclerosis (ALS) and reports suggest possible ASN abnormalities in ALS patients. We assessed ASN immunoreactivity in spinal and brain tissues of subjects who had died of progressive motor neuron disorders (MND). Clinical records of subjects with MND and a comparison group were reviewed to determine the diagnosis according to El-Escariol Criteria of ALS. Cervical, thoracic and lumbar cord sections were stained with an antibody to ASN. A blinded, semiquantitative review of sections from both groups included examination for evidence of spheroids, neuronal staining, cytoplasmic inclusions, anterior horn granules, white and gray matter glial staining, corticospinal tract axonal fiber and myelin changes. MND cases, including ALS and progressive muscular atrophy, displayed significantly increased ASN staining of spheroids ( Pgray and white matter ( P< or =0.05). Significant abnormal staining of corticospinal axon tract fibers and myelin was also observed ( P< or =0.05 and 0.01). Detection of possible ASN-positive neuronal inclusions did not differ between groups. Significant ASN abnormalities were observed in MND. These findings suggest a possible role for ASN in MND; however, the precise nature of this association is unclear.

  13. Impaired baroreflex function in mice overexpressing alpha-synuclein

    Directory of Open Access Journals (Sweden)

    Sheila eFleming

    2013-07-01

    Full Text Available Cardiovascular autonomic dysfunction, such as orthostatic hypotension consequent to baroreflex failure and cardiac sympathetic denervation, is frequently observed in the synucleinopathy Parkinson’s disease (PD. In the present study, the baroreceptor reflex was assessed in mice overexpressing human wildtype alpha-synuclein (Thy1-aSyn, a genetic mouse model of synucleinopathy. The beat-to-beat change in heart rate, computed from R-R interval, in relation to blood pressure was measured in anesthetized and conscious mice equipped with arterial blood pressure telemetry transducers during transient bouts of hypertension and hypotension. Compared to wildtype, tachycardia following nitroprusside-induced hypotension was significantly reduced in Thy1-aSyn mice. Thy1-aSyn mice also showed an abnormal cardiovascular response (i.e., diminished tachycardia to muscarinic blockade with atropine. We conclude that Thy1-aSyn mice have impaired basal and dynamic range of sympathetic and parasympathetic-mediated changes in heart rate and will be a useful model for long-term study of cardiovascular autonomic dysfunction associated with PD.

  14. Age-dependent effects of A53T alpha-synuclein on behavior and dopaminergic function.

    Science.gov (United States)

    Oaks, Adam W; Frankfurt, Maya; Finkelstein, David I; Sidhu, Anita

    2013-01-01

    Expression of A53T mutant human alpha-synuclein under the mouse prion promoter is among the most successful transgenic models of Parkinson's disease. Accumulation of A53T alpha-synuclein causes adult mice to develop severe motor impairment resulting in early death at 8-12 months of age. In younger, pre-symptomatic animals, altered motor activity and anxiety-like behaviors have also been reported. These behavioral changes, which precede severe neuropathology, may stem from non-pathological functions of alpha-synuclein, including modulation of monoamine neurotransmission. Our analysis over the adult life-span of motor activity, anxiety-like, and depressive-like behaviors identifies perturbations both before and after the onset of disease. Young A53T mice had increased distribution of the dopamine transporter (DAT) to the membrane that was associated with increased striatal re-uptake function. DAT function decreased with aging, and was associated with neurochemical alterations that included increased expression of beta-synuclein and gamma synuclein. Prior to normalization of dopamine uptake, transient activation of Tau kinases and hyperphosphorylation of Tau in the striatum were also observed. Aged A53T mice had reduced neuron counts in the substantia nigra pars compacta, yet striatal medium spiny neuron dendritic spine density was largely maintained. These findings highlight the involvement of the synuclein family of proteins and phosphorylation of Tau in the response to dopaminergic dysfunction of the nigrostriatal pathway.

  15. Age-dependent effects of A53T alpha-synuclein on behavior and dopaminergic function.

    Directory of Open Access Journals (Sweden)

    Adam W Oaks

    Full Text Available Expression of A53T mutant human alpha-synuclein under the mouse prion promoter is among the most successful transgenic models of Parkinson's disease. Accumulation of A53T alpha-synuclein causes adult mice to develop severe motor impairment resulting in early death at 8-12 months of age. In younger, pre-symptomatic animals, altered motor activity and anxiety-like behaviors have also been reported. These behavioral changes, which precede severe neuropathology, may stem from non-pathological functions of alpha-synuclein, including modulation of monoamine neurotransmission. Our analysis over the adult life-span of motor activity, anxiety-like, and depressive-like behaviors identifies perturbations both before and after the onset of disease. Young A53T mice had increased distribution of the dopamine transporter (DAT to the membrane that was associated with increased striatal re-uptake function. DAT function decreased with aging, and was associated with neurochemical alterations that included increased expression of beta-synuclein and gamma synuclein. Prior to normalization of dopamine uptake, transient activation of Tau kinases and hyperphosphorylation of Tau in the striatum were also observed. Aged A53T mice had reduced neuron counts in the substantia nigra pars compacta, yet striatal medium spiny neuron dendritic spine density was largely maintained. These findings highlight the involvement of the synuclein family of proteins and phosphorylation of Tau in the response to dopaminergic dysfunction of the nigrostriatal pathway.

  16. Analysis of alpha-synuclein in malignant melanoma - development of a SRM quantification assay.

    Directory of Open Access Journals (Sweden)

    Charlotte Welinder

    Full Text Available Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases. Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment. The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients. A targeted Selected Reaction Monitoring (SRM assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein. Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank. The calibration curve using peak area ratio (heavy/light versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences. In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues. Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.

  17. The Identification of Alpha-Synuclein as the First Parkinson Disease Gene.

    Science.gov (United States)

    Nussbaum, Robert L

    2017-01-01

    In this Commentary, I describe the events that led from an NINDS-sponsored Workshop on Parkinson Disease Research in 1995, where I was asked to speak about the genetics of Parkinson disease, to the identification a mere two years later of a mutation in alpha-synuclein as the cause of autosomal dominant Parkinson disease in the Contursi kindred. I review the steps we took to first map and then find the mutation in the alpha-synuclein locus and describe the obstacles and the role of serendipity in facilitating the work. Although alpha-synuclein mutations are a rare cause of hereditary PD, the importance of this finding goes far beyond the rare families with hereditary disease because it pinpointed alpha-synuclein as a key contributor to the far more common sporadic form of Parkinson disease. This work confirms William Harvey's observation from 350 years ago that studying rarer forms of a disease is an excellent way to understand the more common forms of that disease. The identification of synuclein's role in hereditary Parkinson disease has opened new avenues of research into the pathogenesis and potential treatments of the common form of Parkinson disease that affects many millions of Americans and tens of millions of human beings worldwide.

  18. Alpha-synuclein in peripheral tissues and body fluids as a biomarker for Parkinson's disease - a systematic review.

    Science.gov (United States)

    Malek, N; Swallow, D; Grosset, K A; Anichtchik, O; Spillantini, M; Grosset, D G

    2014-08-01

    Parkinson's disease (PD) is neuropathologically characterized as an alpha-synucleinopathy. Alpha-synuclein-containing inclusions are stained as Lewy bodies and Lewy neurites in the brain, which are the pathological hallmark of PD. However, alpha-synuclein-containing inclusions in PD are not restricted to the central nervous system, but are also found in peripheral tissues. Alpha-synuclein levels can also be measured in body fluids. The aim of this study was to conduct a systematic review of available evidence to determine the utility of alpha-synuclein as a peripheral biomarker of PD. We searched PubMed (1948 to 26 May 2013), Embase (1974 to 26 May 2013), the Cochrane Library (up to 26 May 2013), LILACS (up to 26 May 2013) and CINAHL (up to 26 May 2013) for the studies of alpha-synuclein in peripheral tissues or body fluids in PD. A total of 49 studies fulfilled the search criteria. Peripheral tissues such as colonic mucosa showed a sensitivity of 42-90% and a specificity of 100%; submandibular salivary glands showed sensitivity and specificity of 100%; skin biopsy showed 19% sensitivity and 80% specificity in detecting alpha-synuclein pathology. CSF alpha-synuclein had 71-94% sensitivity and 25-53% specificity for distinguishing PD from controls. Plasma alpha-synuclein had 48-53% sensitivity and 69-85% specificity. Neither plasma nor CSF alpha-synuclein is presently a reliable marker of PD. This differs from alpha-synuclein in solid tissue samples of the enteric and autonomic nervous system, which offer some potential as a surrogate marker of brain synucleinopathy.

  19. Copper binding regulates intracellular alpha-synuclein localisation, aggregation and toxicity.

    Science.gov (United States)

    Wang, Xiaoyan; Moualla, Dima; Wright, Josephine A; Brown, David R

    2010-05-01

    Alpha-synuclein is a natively unfolded protein that aggregates and forms inclusions that are associated with a range of diseases that include Parkinson's Disease and Dementia with Lewy Bodies. The mechanism behind the formation of these inclusions and their possible role in disease remains unclear. Alpha-synuclein has also been shown to bind metals including copper and iron. We used a cell culture model of alpha-synuclein aggregation to examine the relationship between metals and formation of aggregates of the protein. While the levels of iron appear to have no role in aggregate formation or localisation of the protein in cells, copper appears to be important for both aggregation and cellular localisation of alpha-synuclein. Reduction in cellular copper resulted in a great decrease in aggregate formation both in terms of large aggregates visible in cells and oligomers observed in western blot analysis of cell extracts. Reduction in copper also resulted in a change in localisation of the protein which became more intensely localised to the plasma membrane in medium with low copper. These changes were reversed when copper was restored to the cells. Mutants of the copper binding domains altered the response to copper. Deletion of either the N- or C-termini resulted in a loss of aggregation while deletion of the C-termini also resulted in a loss of membrane association. Increased expression of alpha-synuclein also increased cell sensitivity to the toxicity of copper. These results suggest that the potential pathological role of alpha-synuclein aggregates is dependent upon the copper binding capacity of the protein.

  20. Alpha-synuclein in familial Alzheimer disease: epitope mapping parallels dementia with Lewy bodies and Parkinson disease.

    Science.gov (United States)

    Lippa, C F; Schmidt, M L; Lee, V M; Trojanowski, J Q

    2001-11-01

    Alpha-synuclein is a major component of Lewy bodies (LBs) in Parkinson disease and dementia with LBs and of glial cytoplasmic inclusions in multiple system atrophy. However, epitope mapping for alpha-synuclein is distinctive in different neurodegenerative diseases. The reasons for this are poorly understood but may reflect fundamental differences in disease mechanisms. To investigate the alpha-synuclein epitope mapping properties of LBs in familial Alzheimer disease. We compared LBs in familial Alzheimer disease with those in synucleinopathies by probing 6 brains of persons with familial Alzheimer disease using a panel of antibodies to epitopes spanning the alpha-synuclein protein. Results were compared with data from brains of persons with Parkinson disease, dementia with LBs, and multiple system atrophy. The brains of persons with familial Alzheimer disease showed consistent staining of LBs with all antibodies, similar to Parkinson disease and dementia with LBs but different from alpha-synuclein aggregates that occurred in multiple system atrophy. These data suggest that the epitope profiles of alpha-synuclein in LBs are similar, regardless of whether the biological trigger is related to synuclein or a different genetic pathway. These findings support the hypothesis that the mechanism of alpha-synuclein aggregation is the same within cell types but distinctive between cell types.

  1. Features of alpha-synuclein that could explain the progression and irreversibility of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Scarlet eGallegos

    2015-03-01

    Full Text Available Alpha-synuclein is a presynaptic protein expressed throughout the central nervous system, and it is the main component of Lewy bodies, one of the histopathological features of Parkinson’s disease (PD which is a progressive and irreversible neurodegenerative disorder. The conformational flexibility of α-synuclein allows it to adopt different conformations, i.e. bound to membranes or form aggregates, the oligomers are believed to be the more toxic species. In this review, we will focus on two major features of α-synuclein, transmission and toxicity that could help to understand the pathological characteristics of PD. One important feature of α-synuclein is its ability to be transmitted from neuron to neuron using mechanisms such as endocytosis, plasma membrane penetration or through exosomes, thus propagating the Lewy body pathology to different brain regions thereby contributing to the progressiveness of PD. The second feature of α-synuclein is that it confers cytotoxicity to recipient cells, principally when it is in an oligomeric state. This form causes mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, proteasome impairment, disruption of plasma membrane and pore formation, and lead to apoptosis pathway activation and consequent cell death. The complexity of α-synuclein oligomerization and formation of toxic species could be a major factor for the irreversibility of PD and could also explain the lack of successful therapies to halt the disease.

  2. Mechanisms of Alpha-synuclein Aggregation and Toxicity

    Science.gov (United States)

    2004-09-01

    receptor (Pael-R) [14,36]. The interaction of parkin with Brain samples CHIP suggests that it operates in the endoplasmic re- Case PMI Age Sex Diagnosis...that Car2 is significantly oxidized in A30P α- synuclein mice and since oxidative preteen generally have decreased acitivity [35], this result

  3. Observation of multiple intermediates in alpha-synuclein fibril formation by singular value decomposition analysis.

    Science.gov (United States)

    Kamiyoshihara, Tomoaki; Kojima, Masaki; Uéda, Kenji; Tashiro, Mitsuru; Shimotakahara, Sakurako

    2007-04-06

    One of the most well known characteristics for Parkinson's disease (PD) is a polymerization of wild-type or mutant alpha-synuclein into aggregates and fibrils, commonly observed as Lewy bodies and Lewy neuritis in PD patients. Although numerous studies on alpha-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation are not well understood yet. In the present study, structural properties and propensities to form fibrils of wild-type, A30P, E46K, and A53T alpha-synucleins were investigated using fluorescence and circular dichroism (CD) methods. The results from these studies were analyzed using singular value decomposition (SVD) method which estimates a number of conformationally independent species for a given process. The time-dependent CD spectra of the wild-type alpha-synuclein indicated a multi-step process in the fibril formation, and SVD analysis using the time-dependent CD spectra revealed that five or nine intermediates were formed at the early stage of fibrillation.

  4. CSF alpha-synuclein does not discriminate dementia with lewy bodies from Alzheimer's disease.

    NARCIS (Netherlands)

    Reesink, F.E.; Lemstra, A.W.; Dijk, K.D. van; Berendse, H.W.; Berg, W.D. van de; Klein, M.; Blankenstein, M.A.; Scheltens, P.; Verbeek, M.M.; Flier, W.M. van der

    2010-01-01

    In this study, we assessed whether cerebrospinal fluid (CSF) levels of the biomarker alpha-synuclein have a diagnostic value in differential diagnosis of dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). We also analyzed associations between CSF biomarkers and cognitive performance in DL

  5. C-elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

    NARCIS (Netherlands)

    van Ham, Tjakko J.; Thijssen, Karen L.; Breitling, Rainer; Hofstra, Robert M. W.; Plasterk, Ronald H. A.; Nollen, Ellen A. A.

    2008-01-01

    Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of

  6. Increased frequency of alpha-synuclein in the substantia nigra in human immunodeficiency virus infection.

    Science.gov (United States)

    Khanlou, Negar; Moore, David J; Chana, Gursharan; Cherner, Mariana; Lazzaretto, Deborah; Dawes, Sharron; Grant, Igor; Masliah, Eliezer; Everall, Ian P

    2009-04-01

    The frequency of neurodegenerative markers among long surviving human immunodeficiency virus (HIV)-infected individuals is unknown, therefore, the present study investigated the frequency of alpha-synuclein, beta-amyloid, and HIV-associated brain pathology in the brains of older HIV-infected individuals. We examined the substantia nigra of 73 clinically well-characterized HIV-infected individuals aged 50 to 76 years from the National NeuroAIDS Tissue Consortium. We also examined the frontal and temporal cortical regions of a subset of 36 individuals. Neuritic alpha-synuclein expression was found in 16% (12/73) of the substantia nigra of the HIV+cases and none of the older control cases (0/18). beta-Amyloid deposits were prevalent and found in nearly all of the HIV+cases (35/36). Despite these increases of degenerative pathology, HIV-associated brain pathology was present in only 10% of cases. Among older HIV+adults, HIV-associated brain pathology does not appear elevated; however, the frequency of both alpha-synuclein and beta-amyloid is higher than that found in older healthy persons. The increased prevalence of alpha-synuclein and beta-amyloid in the brains of older HIV-infected individuals may predict an increased risk of developing neurodegenerative disease.

  7. The Identification of Alpha-Synuclein as the First Parkinson Disease Gene

    Science.gov (United States)

    Nussbaum, Robert L.

    2017-01-01

    In this Commentary, I describe the events that led from an NINDS-sponsored Workshop on Parkinson Disease Research in 1995, where I was asked to speak about the genetics of Parkinson disease, to the identification a mere two years later of a mutation in alpha-synuclein as the cause of autosomal dominant Parkinson disease in the Contursi kindred. I review the steps we took to first map and then find the mutation in the alpha-synuclein locus and describe the obstacles and the role of serendipity in facilitating the work. Although alpha-synuclein mutations are a rare cause of hereditary PD, the importance of this finding goes far beyond the rare families with hereditary disease because it pinpointed alpha-synuclein as a key contributor to the far more common sporadic form of Parkinson disease. This work confirms William Harvey’s observation from 350 years ago that studying rarer forms of a disease is an excellent way to understand the more common forms of that disease. The identification of synuclein’s role in hereditary Parkinson disease has opened new avenues of research into the pathogenesis and potential treatments of the common form of Parkinson disease that affects many millions of Americans and tens of millions of human beings worldwide. PMID:28282812

  8. Tri- and Pentamethine Cyanine Dyes for Fluorescent Detection of alpha-Synuclein Oligomeric Aggregates

    NARCIS (Netherlands)

    Kovalska, V.B.; Losytskyy, M.Y.; Tolmachev, O.I.; Slominskii, Y.L.; Segers-Nolten, G.M.; Subramaniam, V.; Yarmoluk, S.M.

    2012-01-01

    The pathogenesis of Parkinson's disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of alpha-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligo

  9. Alpha-synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids

    DEFF Research Database (Denmark)

    Golovko, Mikhail Y; Rosenberger, Thad A; Feddersen, Søren

    2007-01-01

    Previously, we demonstrated that ablation of alpha-synuclein (Snca) reduces arachidonate (20:4n-6) turnover in brain phospholipids through modulation of an endoplasmic reticulum-localized acyl-CoA synthetase (Acsl). The effect of Snca ablation on docosahexaenoic acid (22:6n-3) metabolism is unkno...

  10. Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein

    Science.gov (United States)

    Iljina, Marija; Tosatto, Laura; Choi, Minee L.; Sang, Jason C.; Ye, Yu; Hughes, Craig D.; Bryant, Clare E.; Gandhi, Sonia; Klenerman, David

    2016-09-01

    The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.

  11. Structural transitions in the intrinsically disordered Parkinson's protein alpha-synuclein

    Science.gov (United States)

    Eliezer, David

    2013-03-01

    The protein alpha-synuclein is genetically and histopathologically associated with familial and sporadic Parkinson's disease. Although considered to belong to the category of intrinsically disordered proteins for well over a decade, recent reports have suggested that synuclein may actually exist predominantly in a native, well-structured, tetrameric form. Experiments using in-cell NMR, which bypass potential structural perturbations caused by purification protocols, conclusively demonstrate that recombinant synuclein is in fact highly disordered and monomeric. In the presence of membranes, however, the protein undergoes a coil-to-helix transition to adopt several highly helical conformations, which are proposed to mediate both its normal function and its membrane-induced aggregation into amyloid fibrils. Supported by NIH grant R37AG019391

  12. Expression of alpha-synuclein in different brain parts of adult and aged rats.

    Science.gov (United States)

    Adamczyk, A; Solecka, J; Strosznajder, J B

    2005-03-01

    The synucleins are a family of presynaptic proteins that are abundant in neurons and include alpha-, beta, and gamma-synuclein. Alpha-synuclein (ASN) is involved in several neurodegenerative age-related disorders but its relevance in physiological aging is unknown. In the present study we investigated the expression of ASN mRNA and protein in the different brain parts of the adult (4-month-old) and aged (24-month-old) rats by using RT-PCR technique and Western blot, respectively. Our results indicated that mRNA expression and immunoreactivity of ASN is similar in brain cortex, hippocampus and striatum but markedly lower in cerebellum comparing to the other brain parts. Aging lowers ASN mRNA expression in striatum and cerebellum by about 40%. The immunoreactivity of ASN in synaptic plasma membranes (SPM) from aged brain cortex, hippocampus and cerebellum is significantly lower comparing to adult by 39%, 24% and 65%, respectively. Beta-synuclein (BSN) was not changed in aged brain comparing to adult. Age-related alteration of ASN may affect the nerve terminals structure and function.

  13. Oxidative stress-induced posttranslational modifications of alpha-synuclein: specific modification of alpha-synuclein by 4-hydroxy-2-nonenal increases dopaminergic toxicity.

    Science.gov (United States)

    Xiang, Wei; Schlachetzki, Johannes C M; Helling, Stefan; Bussmann, Julia C; Berlinghof, Marvin; Schäffer, Tilman E; Marcus, Katrin; Winkler, Jürgen; Klucken, Jochen; Becker, Cord-Michael

    2013-05-01

    Aggregation and neurotoxicity of misfolded alpha-synuclein (αSyn) are crucial mechanisms for progressive dopaminergic neurodegeneration associated with Parkinson's disease (PD). Posttranslational modifications (PTMs) of αSyn caused by oxidative stress, including modification by 4-hydroxy-2-nonenal (HNE-αSyn), nitration (n-αSyn), and oxidation (o-αSyn), have been implicated to promote oligomerization of αSyn. However, it is yet unclear if these PTMs lead to different types of oligomeric intermediates. Moreover, little is known about which PTM-derived αSyn species exerts toxicity to dopaminergic cells. In this study, we directly compared aggregation characteristics of HNE-αSyn, n-αSyn, and o-αSyn. Generally, all of them promoted αSyn oligomerization. Particularly, HNE-αSyn and n-αSyn were more prone to forming oligomers than unmodified αSyn. Moreover, these PTMs prevented the formation of amyloid-like fibrils, although HNE-αSyn and o-αSyn were able to generate protofibrillar structures. The cellular effects associated with distinct PTMs were studied by exposing modified αSyn to dopaminergic Lund human mesencephalic (LUHMES) neurons. The cellular toxicity of HNE-αSyn was significantly higher than other PTM species. Furthermore, we tested the toxicity of HNE-αSyn in dopaminergic LUHMES cells and other cell types with low tyrosine hydroxylase (TH) expression, and additionally analyzed the loss of TH-immunoreactive cells in HNE-αSyn-treated LUHMES cells. We observed a selective toxicity of HNE-αSyn to neurons with higher TH expression. Further mechanistic studies showed that HNE-modification apparently increased the interaction of extracellular αSyn with neurons. Moreover, exposure of differentiated LUHMES cells to HNE-αSyn triggered the production of intracellular reactive oxygen species, preceding neuronal cell death. Antioxidant treatment effectively protected cells from the damage triggered by HNE-αSyn. Our findings suggest a specific

  14. Abnormal colonic motility in mice overexpressing human wild-type alpha-synuclein.

    Science.gov (United States)

    Wang, Lixin; Fleming, Sheila M; Chesselet, Marie-Françoise; Taché, Yvette

    2008-05-28

    The presynaptic protein alpha-synuclein (alphaSyn) has been implicated in both familial and sporadic forms of Parkinson's disease. We examined whether human alphaSyn-overexpressing mice under Thy1 promoter (Thy1-alphaSyn) display alterations of colonic function. Basal fecal output was decreased in Thy1-alphaSyn mice fed ad libitum. Fasted/refed Thy1-alphaSyn mice had a slower distal colonic transit than the wild-type mice, as monitored by 2.2-fold increase in time to expel an intracolonic bead and 2.9-fold higher colonic fecal content. By contrast, Thy1-alphaSyn mice had an increased fecal response to novelty stress and corticotropin releasing factor injected intraperipherally. These results indicate that Thy1-alphaSyn mice display altered basal and stress-stimulated propulsive colonic motility and will be a useful model to study gut dysfunction associated with Parkinson's disease.

  15. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia.

    Science.gov (United States)

    Zarranz, Juan J; Alegre, Javier; Gómez-Esteban, Juan C; Lezcano, Elena; Ros, Raquel; Ampuero, Israel; Vidal, Lídice; Hoenicka, Janet; Rodriguez, Olga; Atarés, Begoña; Llorens, Verónica; Gomez Tortosa, Estrella; del Ser, Teodoro; Muñoz, David G; de Yebenes, Justo G

    2004-02-01

    Familial parkinsonism and dementia with cortical and subcortical Lewy bodies is uncommon, and no genetic defect has been reported in the previously described sibships. We present a Spanish family with autosomal dominant parkinsonism, dementia, and visual hallucinations of variable severity. The postmortem examination showed atrophy of the substantia nigra, lack of Alzheimer pathology, and numerous Lewy bodies which were immunoreactive to alpha-synuclein and ubiquitin in cortical and subcortical areas. Sequencing of the alpha-synuclein gene showed a novel, nonconservative E46K mutation in heterozygosis. The E46K mutation was present in all affected family members and in three young asymptomatic subjects, but it was absent in healthy and pathological controls. The novel mutation, that substitutes a dicarboxylic amino acid, glutamic acid, with a basic amino acid such as lysine in a much conserved area of the protein, is likely to produce severe disturbance of protein function. Our data show that, in addition to the previously described hereditary alpha-synucleinopathies, dementia with Lewy bodies is related to mutation of alpha-synuclein.

  16. Bioinorganic chemistry of copper coordination to alpha-synuclein: relevance to Parkinson's disease.

    OpenAIRE

    Binolfi, A.; L. Quintanar; Bertoncini, C.; Griesinger, C.; FERNANDEZ, C., LEOZ, J.

    2012-01-01

    Alpha-synuclein (AS) aggregation is associated with neurodegeneration in Parkinson'sdisease (PD). At the same time, alterations in metal ion homeostasis may play a pivotal role in the progression of AS amyloid assembly and the onset of PD. Elucidation of the structural basis directing AS–metal interactions and their effect on AS aggregation constitutes a key step toward understanding the role of metal ions in AS amyloid formation and neurodegeneration. This work provides a comprehensive revie...

  17. The temporal expression pattern of alpha-synuclein modulates olfactory neurogenesis in transgenic mice.

    Directory of Open Access Journals (Sweden)

    Sebastian R Schreglmann

    Full Text Available Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS under the murine Thy1 (mThy1 promoter, a model known to have a particularly high tg expression associated with impaired olfaction.Survival of newly generated neurons (NeuN-positive in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.

  18. Copper(II) binding to alpha-synuclein, the Parkinson's protein.

    Science.gov (United States)

    Lee, Jennifer C; Gray, Harry B; Winkler, Jay R

    2008-06-04

    Variations in tryptophan fluorescence intensities confirm that copper(II) interacts with alpha-synuclein, a protein implicated in Parkinson's disease. Trp4 fluorescence decay kinetics measured for the F4W protein show that Cu(II) binds tightly (Kd 100 nM) near the N-terminus at pH 7. Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site.

  19. Elevated alpha-synuclein impairs innate immune cell function and provides a potential peripheral biomarker for Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Shyra J Gardai

    Full Text Available Alpha-synuclein protein is strongly implicated in the pathogenesis Parkinson's disease. Increased expression of α-synuclein due to genetic multiplication or point mutations leads to early onset disease. While α-synuclein is known to modulate membrane vesicle dynamics, it is not clear if this activity is involved in the pathogenic process or if measurable physiological effects of α-synuclein over-expression or mutation exist in vivo. Macrophages and microglia isolated from BAC α-synuclein transgenic mice, which overexpress α-synuclein under regulation of its own promoter, express α-synuclein and exhibit impaired cytokine release and phagocytosis. These processes were affected in vivo as well, both in peritoneal macrophages and microglia in the CNS. Extending these findings to humans, we found similar results with monocytes and fibroblasts isolated from idiopathic or familial Parkinson's disease patients compared to age-matched controls. In summary, this paper provides 1 a new animal model to measure α-synuclein dysfunction; 2 a cellular system to measure synchronized mobilization of α-synuclein and its functional interactions; 3 observations regarding a potential role for innate immune cell function in the development and progression of Parkinson's disease and other human synucleinopathies; 4 putative peripheral biomarkers to study and track these processes in human subjects. While altered neuronal function is a primary issue in PD, the widespread consequence of abnormal α-synuclein expression in other cell types, including immune cells, could play an important role in the neurodegenerative progression of PD and other synucleinopathies. Moreover, increased α-synuclein and altered phagocytosis may provide a useful biomarker for human PD.

  20. Granular assembly of alpha-synuclein leading to the accelerated amyloid fibril formation with shear stress.

    Science.gov (United States)

    Bhak, Ghibom; Lee, Jung-Ho; Hahn, Ji-Sook; Paik, Seung R

    2009-01-01

    alpha-Synuclein participates in the Lewy body formation of Parkinson's disease. Elucidation of the underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop a controlling strategy toward the disease, but also to apply the protein fibrils for future biotechnology. Discernable homogeneous granules of alpha-synuclein composed of approximately 11 monomers in average were isolated in the middle of a lag phase during the in vitro fibrillation process. They were demonstrated to experience almost instantaneous fibrillation during a single 12-min centrifugal membrane-filtration at 14,000 x g. The granular assembly leading to the drastically accelerated fibril formation was demonstrated to be a result of the physical influence of shear force imposed on the preformed granular structures by either centrifugal filtration or rheometer. Structural rearrangement of the preformed oligomomeric structures is attributable for the suprastructure formation in which the granules act as a growing unit for the fibril formation. To parallel the prevailing notion of nucleation-dependent amyloidosis, we propose a double-concerted fibrillation model as one of the mechanisms to explain the in vitro fibrillation of alpha-synuclein, in which two consecutive concerted associations of monomers and subsequent oligomeric granular species are responsible for the eventual amyloid fibril formation.

  1. Granular assembly of alpha-synuclein leading to the accelerated amyloid fibril formation with shear stress.

    Directory of Open Access Journals (Sweden)

    Ghibom Bhak

    Full Text Available alpha-Synuclein participates in the Lewy body formation of Parkinson's disease. Elucidation of the underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop a controlling strategy toward the disease, but also to apply the protein fibrils for future biotechnology. Discernable homogeneous granules of alpha-synuclein composed of approximately 11 monomers in average were isolated in the middle of a lag phase during the in vitro fibrillation process. They were demonstrated to experience almost instantaneous fibrillation during a single 12-min centrifugal membrane-filtration at 14,000 x g. The granular assembly leading to the drastically accelerated fibril formation was demonstrated to be a result of the physical influence of shear force imposed on the preformed granular structures by either centrifugal filtration or rheometer. Structural rearrangement of the preformed oligomomeric structures is attributable for the suprastructure formation in which the granules act as a growing unit for the fibril formation. To parallel the prevailing notion of nucleation-dependent amyloidosis, we propose a double-concerted fibrillation model as one of the mechanisms to explain the in vitro fibrillation of alpha-synuclein, in which two consecutive concerted associations of monomers and subsequent oligomeric granular species are responsible for the eventual amyloid fibril formation.

  2. Melatonin inhibits maneb-induced aggregation of alpha-synuclein in rat pheochromocytoma cells.

    Science.gov (United States)

    Ishido, Masami

    2007-03-01

    Melatonin, a secretory product of the pineal gland, is involved in the regulation of circadian and seasonal rhythms, in oncostasis, and in inducing osteoblast differentiation. Furthermore, melatonin is a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. In this study, the antioxidant nature of melatonin was shown to prevent cultured neural cells from apoptosis induced by endocrine-disrupting chemical, maneb. The neurotoxicity of the fungicide, maneb (1 microg/mL), on the PC12 cells was elicited through apoptotic cell death, concomitant with aggregation of alpha-synuclein, a feature of Parkinson's disease. Activation of caspase-3/7 was associated with this process. A fluorescence rationing technique using a mitochondrial dye revealed that maneb altered the mitochondrial membrane potential of the neural cells. However, melatonin (1 nm) largely prevented the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. Furthermore, aggregation of alpha-synuclein by maneb was also inhibited by melatonin. Thus, melatonin prevents maneb-induced neurodegeneration at a nighttime physiological blood concentration, most likely by inhibiting the aggregation of alpha-synuclein as well as preventing mitochondrial dysfunction in PC 12 cells.

  3. Domain a' of protein disulfide isomerase plays key role in inhibiting alpha-synuclein fibril formation.

    Science.gov (United States)

    Cheng, Han; Wang, Lei; Wang, Chih-chen

    2010-07-01

    alpha-Synuclein (alpha Syn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to alpha Syn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits alpha Syn fibril formation in vitro. In PDI molecule with a structure of abb'xa'c, domain a' was found to be essential and sufficient for PDI to inhibit alpha Syn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during alpha Syn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.

  4. Dynamic transport and localization of alpha-synuclein in primary hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Woods Wendy S

    2010-02-01

    Full Text Available Abstract Background Alpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis. Abnormal accumulation of α-synuclein aggregates in dopaminergic neurons of the substantia nigra is diagnostic of sporadic Parkinson's disease, and mutations in the protein are linked to early onset forms of the disease. The folded conformation of the protein varies depending upon its environment and other factors that are poorly understood. When bound to phospholipid membranes, α-synuclein adopts a helical conformation that mediates specific interactions with other proteins. Results To investigate the role of the helical domain in transport and localization of α-synuclein, eGFP-tagged constructs were transfected into rat primary hippocampal neurons at 7 DIV. A series of constructs were analyzed in which each individual exon was deleted, for comparison to previous studies of lipid affinity and α-helix content. A53T and A30P substitutions, representing Parkinson's disease-associated variants, were analyzed as well. Single exon deletions within the lipid-binding N-terminal domain of α-synuclein (exons 2, 3, and 4 partially disrupted its presynaptic localization at 17-21 DIV, resulting in increased diffuse labeling of axons. Similar results were obtained for A30P, which exhibits decreased lipid binding, but not A53T. To examine whether differences in presynaptic enrichment were related to deficiencies in transport velocity, transport was visualized via live cell microscopy. Tagged α-synuclein migrated at a rate of 1.85 ± 0.09 μm/s, consistent with previous reports, and single exon deletion mutants migrated at similar rates, as did A30P. Deletion of the entire N-terminal lipid-binding domain (Δ234GFP did not significantly alter rates of particle movement, but decreased the number of moving particles. Only the A53TGFP mutant exhibited a significant decrease in transport velocity as compared to ASGFP. Conclusions

  5. Identification of the minimal copper(II)-binding alpha-synuclein sequence.

    Science.gov (United States)

    Jackson, Mark S; Lee, Jennifer C

    2009-10-05

    Parkinson's disease has been long linked to environmental factors, such as transition metals and recently to alpha-synuclein, a presynaptic protein. Using tryptophan-containing peptides, we identified the minimal Cu(II)-binding sequence to be within the first four residues, MDV(F/W), anchored by the alpha-amino terminus. In addition, mutant peptide 1-10 (Lys --> Arg) verified that neither Lys6 nor Lys10 are necessary for Cu(II) binding. Interestingly, Trp4 excited-state decay kinetics measured for peptides and proteins reveal two quenching modes, possibly arising from two distinct Cu(II)-polypeptide structures.

  6. Interactions between Calcium and Alpha-Synuclein in Neurodegeneration

    Science.gov (United States)

    Rcom-H’cheo-Gauthier, Alex; Goodwin, Jacob; Pountney, Dean L.

    2014-01-01

    In Parkinson’s disease and some atypical Parkinson’s syndromes, aggregation of the α-synuclein protein (α-syn) has been linked to neurodegeneration. Many triggers for pathological α-syn aggregation have been identified, including port-translational modifications, oxidative stress and raised metal ions, such as Ca2+. Recently, it has been found using cell culture models that transient increases of intracellular Ca2+ induce cytoplasmic α-syn aggregates. Ca2+-dependent α-syn aggregation could be blocked by the Ca2+ buffering agent, BAPTA-AM, or by the Ca2+ channel blocker, Trimethadione. Furthermore, a greater proportion of cells positive for aggregates occurred when both raised Ca2+ and oxidative stress were combined, indicating that Ca2+ and oxidative stress cooperatively promote α-syn aggregation. Current on-going work using a unilateral mouse lesion model of Parkinson’s disease shows a greater proportion of calbindin-positive neurons survive the lesion, with intracellular α-syn aggregates almost exclusively occurring in calbindin-negative neurons. These and other recent findings are reviewed in the context of neurodegenerative pathologies and suggest an association between raised Ca2+, α-syn aggregation and neurotoxicity. PMID:25256602

  7. Interactions between Calcium and Alpha-Synuclein in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Alex Rcom-H'cheo-Gauthier

    2014-08-01

    Full Text Available In Parkinson’s disease and some atypical Parkinson’s syndromes, aggregation of the α-synuclein protein (α-syn has been linked to neurodegeneration. Many triggers for pathological α-syn aggregation have been identified, including port-translational modifications, oxidative stress and raised metal ions, such as Ca2+. Recently, it has been found using cell culture models that transient increases of intracellular Ca2+ induce cytoplasmic α-syn aggregates. Ca2+-dependent α-syn aggregation could be blocked by the Ca2+ buffering agent, BAPTA-AM, or by the Ca2+ channel blocker, Trimethadione. Furthermore, a greater proportion of cells positive for aggregates occurred when both raised Ca2+ and oxidative stress were combined, indicating that Ca2+ and oxidative stress cooperatively promote α-syn aggregation. Current on-going work using a unilateral mouse lesion model of Parkinson’s disease shows a greater proportion of calbindin-positive neurons survive the lesion, with intracellular α-syn aggregates almost exclusively occurring in calbindin-negative neurons. These and other recent findings are reviewed in the context of neurodegenerative pathologies and suggest an association between raised Ca2+, α-syn aggregation and neurotoxicity.

  8. Evidence for copper-dioxygen reactivity during alpha-synuclein fibril formation.

    Science.gov (United States)

    Lucas, Heather R; Debeer, Serena; Hong, Myoung-Soon; Lee, Jennifer C

    2010-05-19

    Alpha-synuclein (alpha-syn), a presynaptic protein implicated in Parkinson's disease, binds copper(II) ion (1:1) with submicromolar affinity in vitro. Insights on the molecular details of soluble- and fibrillar-Cu-alpha-syn are gained through X-ray absorption spectroscopy. Our results indicate that the copper coordination environment (3-to-4 N/O ligands, average Cu-ligand distance approximately 1.96 A) exhibits little structural rearrangement upon amyloid formation in spite of the overall polypeptide conformational change from disordered-to-beta-sheet. Interestingly, we find that some population of Cu(II)-alpha-syn reduces to Cu(I)-alpha-syn in the absence of O(2). This autoreduction event appears diminished in the presence of O(2) suggestive of preceding Cu(I)/O(2) chemistry. Evidence for generation of reactive oxygen species is obtained by the observation of new emission features attributed to dityrosine cross-links in fibrillar samples.

  9. Alpha-synuclein lesions in normal aging, Parkinson disease, and Alzheimer disease: evidence from the Baltimore Longitudinal Study of Aging (BLSA).

    Science.gov (United States)

    Mikolaenko, Irina; Pletnikova, Olga; Kawas, Claudia H; O'Brien, Richard; Resnick, Susan M; Crain, Barbara; Troncoso, Juan C

    2005-02-01

    Alpha-synuclein (alpha-synuclein) lesions are characteristic of idiopathic Parkinson disease (PD) and other alpha-synucleinopathies. To study the frequency of alpha-synuclein lesions in normal aging and how frequently they coexist with lesions of Alzheimer disease (AD), we examined the autopsy brains from normal and demented subjects in the Baltimore Longitudinal Study of Aging (BLSA) (n = 117). We found that the overall frequency of alpha-synuclein lesions was 25%, with 100% in 7 cases of PD, 31.5% in 56 cases with AD lesions, and 8.3% among 36 older control brains. Among brains with AD lesions, the frequency of alpha-synuclein pathology was higher in those with higher scores for neuritic plaques, but not in those with higher scores for neurofibrillary tangles. Our observations indicate that alpha-synuclein lesions are uncommon in aged control subjects. Finally, the coexistence of Abeta amyloid and alpha-synuclein pathology in AD brains suggests that the pathogenic mechanism/s leading to the accumulation of Abeta and alpha-synuclein may be similar.

  10. Neuropathology in mice expressing mouse alpha-synuclein.

    Directory of Open Access Journals (Sweden)

    Claus Rieker

    Full Text Available α-Synuclein (αSN in human is tightly linked both neuropathologically and genetically to Parkinson's disease (PD and related disorders. Disease-causing properties in vivo of the wildtype mouse ortholog (mαSN, which carries a threonine at position 53 like the A53T human mutant version that is genetically linked to PD, were never reported. To this end we generated mouse lines that express mαSN in central neurons at levels reaching up to six-fold compared to endogenous mαSN. Unlike transgenic mice expressing human wildtype or mutant forms of αSN, these mαSN transgenic mice showed pronounced ubiquitin immunopathology in spinal cord and brainstem. Isoelectric separation of mαSN species revealed multiple isoforms including two Ser129-phosphorylated species in the most severely affected brain regions. Neuronal Ser129-phosphorylated αSN occurred in granular and small fibrillar aggregates and pathological staining patterns in neurites occasionally revealed a striking ladder of small alternating segments staining either for Ser129-phosphorylated αSN or ubiquitin but not both. Axonal degeneration in long white matter tracts of the spinal cord, with breakdown of myelin sheaths and degeneration of neuromuscular junctions with loss of integrity of the presynaptic neurofilament network in mαSN transgenic mice, was similar to what we have reported for mice expressing human αSN wildtype or mutant forms. In hippocampal neurons, the mαSN protein accumulated and was phosphorylated but these neurons showed no ubiquitin immunopathology. In contrast to the early-onset motor abnormalities and muscle weakness observed in mice expressing human αSN, mαSN transgenic mice displayed only end-stage phenotypic alterations that manifested alongside with neuropathology. Altogether these findings show that increased levels of wildtype mαSN does not induce early-onset behavior changes, but drives end-stage pathophysiological changes in murine neurons that are

  11. More than just two peas in a pod: common amyloidogenic properties of tau and alpha-synuclein in neurodegenerative diseases.

    Science.gov (United States)

    Lee, Virginia M-Y; Giasson, Benoit I; Trojanowski, John Q

    2004-03-01

    Intracytoplasmic filamentous aggregates, such as neurofibrillary tangles in Alzheimer's disease and Lewy bodies in Parkinson's disease, are composed of the proteins tau and alpha-synuclein, respectively. These pathological inclusions are linked directly to the etiology and mechanisms of disease in a wide spectrum of neurodegenerative disorders, termed 'tauopathies' and 'synucleinopathies'. Emerging evidence indicates that there is frequent overlap of the pathological and clinical features of patients with tauopathies and synucleinopathies, thereby re-enforcing the notion that these disorders might be linked mechanistically. Indeed, several lines of investigation suggest that tau and alpha-synuclein might constitute a unique class of unstructured proteins that assemble predominantly into homopolymeric (rather than heteropolymeric) fibrils, which deposit mainly in separate amyloid inclusions, but occasionally deposit together. Thus, the ability of tau and alpha-synuclein to affect each other directly or indirectly might contribute to the overlap in the clinical and pathological features of tauopathies and synucleinopathies.

  12. Alpha-synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae.

    Science.gov (United States)

    Soper, James H; Roy, Subhojit; Stieber, Anna; Lee, Eliza; Wilson, Robert B; Trojanowski, John Q; Burd, Christopher G; Lee, Virginia M-Y

    2008-03-01

    Aggregated alpha-synuclein (alpha-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, alpha-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human alpha-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of yeast alpha-syn accumulations and their relevance to human synucleinopathies are unknown. Here we provide ultrastructural evidence that alpha-syn accumulations are not comprised of LB-like fibrils, but are associated with clusters of vesicles. Live-cell imaging showed alpha-syn initially localized to the plasma membrane and subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of alpha-syn revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because vesicular clustering is also found in LB-containing neurons of PD brains, alpha-syn-mediated vesicular accumulation in yeast represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies.

  13. Novel Epigenetic Regulation of Alpha-Synuclein Expression in Down Syndrome.

    Science.gov (United States)

    Ramakrishna, Narayan; Meeker, Harry C; Brown, W Ted

    2016-01-01

    Alpha-synuclein (SNCA), a presynaptic protein, is significantly reduced in individuals with Down syndrome (DS) and Ts65Dn mice, a mouse model of DS. Methylation analyses of promoter proximal CpG sites indicate similar reduction in Ts65Dn mice compared to control mice. Epigallocatechin-3-gallate (EGCG), a polyphenolic catechin present in green tea extract, increases methylation of SNCA promoter proximal CpG sites and expression in Ts65Dn mice. These results suggest a positive link between CpG methylation and SNCA expression in Down syndrome.

  14. Dieldrin induces ubiquitin-proteasome dysfunction in alpha-synuclein overexpressing dopaminergic neuronal cells and enhances susceptibility to apoptotic cell death.

    Science.gov (United States)

    Sun, Faneng; Anantharam, Vellareddy; Latchoumycandane, Calivarathan; Kanthasamy, Arthi; Kanthasamy, Anumantha G

    2005-10-01

    Exposure to pesticides is implicated in the etiopathogenesis of Parkinson's disease (PD). The organochlorine pesticide dieldrin is one of the environmental chemicals potentially linked to PD. Because recent evidence indicates that abnormal accumulation and aggregation of alpha-synuclein and ubiquitin-proteasome system dysfunction can contribute to the degenerative processes of PD, in the present study we examined whether the environmental pesticide dieldrin impairs proteasomal function and subsequently promotes apoptotic cell death in rat mesencephalic dopaminergic neuronal cells overexpressing human alpha-synuclein. Overexpression of wild-type alpha-synuclein significantly reduced the proteasomal activity. Dieldrin exposure dose-dependently (0-70 microM) decreased proteasomal activity, and 30 microM dieldrin inhibited activity by more than 60% in alpha-synuclein cells. Confocal microscopic analysis of dieldrin-treated alpha-synuclein cells revealed that alpha-synuclein-positive protein aggregates colocalized with ubiquitin protein. Further characterization of the aggregates with the autophagosomal marker mondansyl cadaverine and the lysosomal marker and dot-blot analysis revealed that these protein oligomeric aggregates were distinct from autophagosomes and lysosomes. The dieldrin-induced proteasomal dysfunction in alpha-synuclein cells was also confirmed by significant accumulation of ubiquitin protein conjugates in the detergent-insoluble fraction. We found that proteasomal inhibition preceded cell death after dieldrin treatment and that alpha-synuclein cells were more sensitive than vector cells to the toxicity. Furthermore, measurement of caspase-3 and DNA fragmentation confirmed the enhanced sensitivity of alpha-synuclein cells to dieldrin-induced apoptosis. Together, our results suggest that increased expression of alpha-synuclein predisposes dopaminergic cells to proteasomal dysfunction, which can be further exacerbated by environmental exposure to certain

  15. Specific fluorescent detection of fibrillar alpha-synuclein using mono- and trimethine cyanine dyes.

    Science.gov (United States)

    Volkova, K D; Kovalska, V B; Balanda, A O; Losytskyy, M Yu; Golub, A G; Vermeij, R J; Subramaniam, V; Tolmachev, O I; Yarmoluk, S M

    2008-02-01

    With the aim of searching of novel amyloid-specific fluorescent probes the ability of series of mono- and trimethine cyanines based on benzothiazole, pyridine and quinoline heterocycle end groups to recognize fibrillar formations of alpha-synuclein (ASN) was studied. For the first time it was revealed that monomethine cyanines can specifically increase their fluorescence in aggregated ASN presence. Dialkylamino-substituted monomethine cyanine T-284 and meso-ethyl-substituted trimethine cyanine SH-516 demonstrated the higher emission intensity and selectivity to aggregated ASN than classic amyloid stain Thioflavin T, and could be proposed as novel efficient fluorescent probes for fibrillar ASN detection. Studies of structure-function dependences have shown that incorporation of amino- or diethylamino- substituents into the 6-position of the benzothiazole heterocycle yields in a appearance of a selective fluorescent response to fibrillar alpha-synuclein presence. Performed calculations of molecular dimensions of studied cyanine dyes gave us the possibility to presume, that dyes bind with their long axes parallel to the fibril axis via insertion into the neat rows (so called 'channels') running along fibril.

  16. Nuclear and neuritic distribution of serine-129 phosphorylated alpha-synuclein in transgenic mice.

    Science.gov (United States)

    Schell, H; Hasegawa, T; Neumann, M; Kahle, P J

    2009-06-02

    Parkinson's disease and dementia with Lewy bodies are very frequent neurological disorders of the elderly. Mutations in the alpha-synuclein (alphaSYN) gene cause Parkinson's disease, often associated with dementia. Neuropathologically these diseases are characterized by the presence of Lewy bodies and Lewy neurites, intraneuronal inclusions mostly composed of alphaSYN protein fibrils. Moreover, alphaSYN is phosphorylated at S129 (phospho-serine-129 [PSer129]) in neuropathological lesions. Using our (Thy1)-[A30P]alphaSYN transgenic mouse model that develops age-dependent impairment in fear conditioning behavior, we investigated PSer129 immunostaining in the brain. We found distinct staining patterns using new, sensitive monoclonal antibodies. Somal and nuclear PSer129 immunoreactivity increased with age in hippocampal and cortical areas as well as the lateral/basolateral amygdalar nuclei and was present also in young, pre-symptomatic mice, but not wild-type controls. The tendency of PSer129 immunostaining to accumulate in the nucleus was confirmed in cell culture. (Thy1)-[A30P]alphaSYN transgenic mice further developed age-dependent, specific neuritic/terminal alphaSYN pathology in the medial parts of the central amygdalar nucleus and one of its projection areas, the lateral hypothalamus. Interestingly, this type of PSer129 neuropathology was thioflavine S negative, unlike the Lewy-like neuropathology present in the brain stem of (Thy1)-[A30P]alphaSYN mice. Thus, alphaSYN becomes phosphorylated in distinct parts of the brain in this alpha-synucleinopathy mouse model, showing age-dependent increases of nuclear PSer129 in cortical brain areas and the formation of neuritic/terminal PSer129 neuropathology with variable amyloid quality within the fear conditioning circuitry and the brain stem.

  17. Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants

    Science.gov (United States)

    Tosatto, Laura; Horrocks, Mathew H.; Dear, Alexander J.; Knowles, Tuomas P. J.; Dalla Serra, Mauro; Cremades, Nunilo; Dobson, Christopher M.; Klenerman, David

    2015-11-01

    Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson’s disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.

  18. Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

    DEFF Research Database (Denmark)

    Kruse, N.; Persson, S.; Alcolea, D.

    2015-01-01

    Decreased levels of alpha-synuclein (aSyn) in cerebrospinal fluid (CSF) in Parkinson's disease and related synucleinopathies have been reported, however, not consistently in all cross-sectional studies. To test the performance of one recently released human-specific enzyme-linked immunosorbent...

  19. Mapping the subcellular distribution of alpha-synuclein in neurons using genetically encoded probes for correlated light and electron microscopy: Implications for Parkinson’s disease pathogenesis

    Science.gov (United States)

    Boassa, D.; Berlanga, M.L.; Yang, M.-L.; Terada, M.; Hu, J.; Bushong, E.A.; Hwang, M.; Masliah, E.; George, J.M.; Ellisman, M.H.

    2013-01-01

    Modifications to the gene encoding human alpha-synuclein have been linked to development of Parkinson’s disease. The highly conserved structure of alpha-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human alpha-synuclein including new genetic tags developed for correlated LM and EM (the tetracysteine-biarsenical labeling system or the new fluorescent protein for EM, MiniSOG), we determined the distribution of alpha-synuclein when over-expressed in primary neurons at supramolecular and cellular scales, in three dimensions (3D). We observed specific association of alpha-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, alpha-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice over-expressing human alpha-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. 3D electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulo-vesicular structures similar to what observed in vitro. We propose that alpha-synuclein over-expression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that alpha- synuclein is involved in processes associated with the sorting, channeling, packaging and transport of synaptic material destined for degradation. PMID:23392688

  20. Conformational equilibria in monomeric alpha-synuclein at the single-molecule level.

    Directory of Open Access Journals (Sweden)

    Massimo Sandal

    2008-01-01

    Full Text Available Human alpha-Synuclein (alphaSyn is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of alphaSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between alphaSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy-based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant alphaSyn. The conformational heterogeneity of monomeric alphaSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and "beta-like" structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the "beta-like" structures significantly increased in different conditions promoting the aggregation of alphaSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric alphaSyn conformers, positively correlated with conditions known to promote the formation of

  1. Nigrostriatal dynein changes in A53T alpha-synuclein transgenic mice [v1; ref status: indexed, http://f1000r.es/2wb

    Directory of Open Access Journals (Sweden)

    Yan Liu

    2014-03-01

    Full Text Available The accumulation of misfolded a-synuclein is mechanistically linked to neurodegeneration in Parkinson’s disease (PD and other alpha-synucleinopathies. However, how alpha-synuclein causes neurodegeneration is unresolved. Several studies have supported the involvement of dynein, the major motor for retrograde axonal transport in alpha-synuclein-dependent neurodegeneration, especially in the nigrostriatal system. Therefore, we examined the nigrostriatal dyneins in transgenic mice that overexpress human A53T alpha-synuclein and recapitulate key features of a PD-like neuronal synucleinopathy. Age-matched nontransgenic littermates were used as controls. The results demonstrated that the protein level of dynein was decreased in the striatum, whereas it was elevated in the substantia nigra. Double immunostaining results revealed that the reduction in dynein level was associated with aggregation of A53T a-synuclein in the striatum. Furthermore, we performed a quantitative analysis of motor behaviors in A53T alpha-synuclein transgenic mice and controls using a modified open field test. We demonstrated that the protein level of dynein in the striatum was significantly correlated with the motor behaviors. Together, our data indicate that dynein changes in the nigrostriatal system of A53T alpha-synuclein transgenic mice may contribute to their severe movement disorder.

  2. Targeted overexpression of human alpha-synuclein in oligodendroglia induces lesions linked to MSA-like progressive autonomic failure.

    Science.gov (United States)

    Stemberger, Sylvia; Poewe, Werner; Wenning, Gregor K; Stefanova, Nadia

    2010-08-01

    Multiple system atrophy (MSA) is a rare neurodegenerative disease of undetermined cause manifesting with progressive autonomic failure (AF), cerebellar ataxia and parkinsonism due to neuronal loss in multiple brain areas associated with (oligodendro)glial cytoplasmic alpha-synuclein (alpha SYN) inclusions (GCIs). Using proteolipid protein (PLP)-alpha-synuclein (alpha SYN) transgenic mice we have previously reported parkinsonian motor deficits triggered by MSA-like alpha SYN inclusions. We now extend these observations by demonstrating degeneration of brain areas that are closely linked to progressive AF and other non-motor symptoms in MSA, in (PLP)-alpha SYN transgenic mice as compared to age-matched non-transgenic controls. We show delayed loss of cholinergic neurons in nucleus ambiguus at 12 months of age as well as early neuronal loss in laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus and Onuf's nucleus at 2 months of age associated with alpha SYN oligodendroglial overexpression. We also report that neuronal loss triggered by MSA-like alpha SYN inclusions is absent up to 12 months of age in the thoracic intermediolateral cell column suggesting a differential dynamic modulation of alpha SYN toxicity within the murine autonomic nervous system. Although the spatial and temporal evolution of central autonomic pathology in MSA is unknown our findings corroborate the utility of the (PLP)-alpha SYN transgenic mouse model as a testbed for the study of oligodendroglial alpha SYN mediated neurodegeneration replicating both motor and non-motor aspects of MSA.

  3. CpG demethylation enhances alpha-synuclein expression and affects the pathogenesis of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Lumine Matsumoto

    Full Text Available BACKGROUND: Alpha-synuclein (SNCA gene expression is an important factor in the pathogenesis of Parkinson's disease (PD. Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD. CpG methylation in the promoter region may also influence SNCA expression. METHODOLOGY/PRINCIPAL FINDINGS: By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression. Postmortem brain analysis revealed regional non-specific methylation differences in this CpG region in the anterior cingulate and putamen among controls and PD; however, in the substantia nigra of PD, methylation was significantly decreased. CONCLUSIONS/SIGNIFICANCE: This CpG region may function as an intronic regulatory element for SNCA gene. Our findings suggest that a novel epigenetic regulatory mechanism controlling SNCA expression influences PD pathogenesis.

  4. Aggregation of alpha-synuclein by a coarse-grained Monte Carlo simulation

    Science.gov (United States)

    Farmer, Barry; Pandey, Ras

    Alpha-synuclein, an intrinsic protein abundant in neurons, is believed to be a major cause of neurodegenerative diseases (e.g. Alzheimer, Parkinson's disease). Abnormal aggregation of ASN leads to Lewy bodies with specific morphologies. We investigate the self-organizing structures in a crowded environment of ASN proteins by a coarse-grained Monte Carlo simulation. ASN is a chain of 140 residues. Structure detail of residues is neglected but its specificity is captured via unique knowledge-based residue-residue interactions. Large-scale simulations are performed to analyze a number local and global physical quantities (e.g. mobility profile, contact map, radius of gyration, structure factor) as a function of temperature and protein concentration. Trend in multi-scale structural variations of the protein in a crowded environment is compared with that of a free protein chain.

  5. Homologous HSV1 and alpha-synuclein peptides stimulate a T cell response in Parkinson's disease.

    Science.gov (United States)

    Caggiu, E; Paulus, K; Galleri, G; Arru, G; Manetti, R; Sechi, G P; Sechi, L A

    2017-09-15

    Environmental factors are implicated in the development of Parkinson's disease (PD). The aim of this study is to investigate the role of cell-mediated immunity upon a specific immune-stimulation with HSV-1 and human alpha-synuclein homologues peptides by using the intracellular cytokine method on Parkinson's patients and healthy controls. The study showed, for the first time, a specific response to TNF-α CD8, CD4 and NK cells after stimulation in PD patients. Our data show a possible role of the immune system in the pathogenesis of Parkinson's disease, and that HSV-1 infections may lead to a progression of the disease. Copyright © 2017. Published by Elsevier B.V.

  6. Conformational equilibria in monomeric alpha-synuclein at the single molecule level

    CERN Document Server

    Sandal, Massimo; Tessari, Isabella; Mammi, Stefano; Bergantino, Elisabetta; Musiani, Francesco; Brucale, Marco; Bubacco, Luigi; Samori', Bruno

    2007-01-01

    Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the aSyn sample, including disordered and "beta-like" structures. We found that this class of "beta-like" structures is directly related to aSyn ag...

  7. Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes.

    Science.gov (United States)

    Colasanti, T; Vomero, M; Alessandri, C; Barbati, C; Maselli, A; Camperio, C; Conti, F; Tinari, A; Carlo-Stella, C; Tuosto, L; Benincasa, D; Valesini, G; Malorni, W; Pierdominici, M; Ortona, E

    2014-05-29

    It has been demonstrated that α-synuclein can aggregate and contribute to the pathogenesis of some neurodegenerative diseases and it is capable of hindering autophagy in neuronal cells. Here, we investigated the implication of α-synuclein in the autophagy process in primary human T lymphocytes. We provide evidence that: (i) knocking down of the α-synuclein gene resulted in increased autophagy, (ii) autophagy induction by energy deprivation was associated with a significant decrease of α-synuclein levels, (iii) autophagy inhibition by 3-methyladenine or by ATG5 knocking down led to a significant increase of α-synuclein levels, and (iv) autophagy impairment, constitutive in T lymphocytes from patients with systemic lupus erythematosus, was associated with abnormal accumulation of α-synuclein aggregates. These results suggest that α-synuclein could be considered as an autophagy-related marker of peripheral blood lymphocytes, potentially suitable for use in the clinical practice.

  8. Rab1A over-expression prevents Golgi apparatus fragmentation and partially corrects motor deficits in an alpha-synuclein based rat model of Parkinson's disease.

    Science.gov (United States)

    Coune, P G; Bensadoun, J C; Aebischer, P; Schneider, B L

    2011-01-01

    Although the overabundance of human alpha-synuclein in nigral dopaminergic neurons is considered to play a pathogenic role in Parkinson's disease (PD), it remains unclear how alpha-synuclein leads to neuronal degeneration and motor symptoms. Here, we explored the effect of human alpha-synuclein in the rat substantia nigra following AAV-mediated gene delivery inducing a moderate loss of dopaminergic neurons together with motor impairments. A significant fraction of the surviving nigral neurons were found to express human αSyn and displayed a pathological fragmentation of the Golgi apparatus. This observation prompted further investigation on the role of the secretory pathway, in particular at the ER/Golgi level, in alpha-synuclein toxicity. To address this question, we co-expressed human alpha-synuclein with Rab1A, a regulator of ER-to-Golgi vesicular trafficking, and found a significant reduction of Golgi fragmentation. Rab1A did not protect the dopaminergic neurons from the alpha-synuclein-induced degeneration that occurred within several months following vector injection. However, we observed in animals co-expressing Rab1A an improvement of motor behavior that correlates with the rescue of normal Golgi morphology in alpha-synuclein-expressing dopaminergic neurons. The non-prenylable mutant Rab1A-DeltaCC did not produce any of the effects observed with the wild-type form of Rab1A, linking the protective role of Rab1A with its activity in ER-to-Golgi vesicular trafficking. In conclusion, Rab1A can rescue the Golgi fragmentation caused by the overabundance of alpha-synuclein in nigral dopaminergic neurons, improving the ability of the surviving neurons to control motor function in hemiparkinsonian animals.

  9. A Swedish family with de novo alpha-synuclein A53T mutation: evidence for early cortical dysfunction

    DEFF Research Database (Denmark)

    Puschmann, Andreas; Ross, Owen A; Vilariño-Güell, Carles;

    2009-01-01

    A de novo alpha-synuclein A53T (p.Ala53 Th; c.209G > A) mutation has been identified in a Swedish family with autosomal dominant Parkinson's disease (PD). Two affected individuals had early-onset (before 31 and 40 years), severe levodopa-responsive PD with prominent dysphasia, dysarthria, and cog......A de novo alpha-synuclein A53T (p.Ala53 Th; c.209G > A) mutation has been identified in a Swedish family with autosomal dominant Parkinson's disease (PD). Two affected individuals had early-onset (before 31 and 40 years), severe levodopa-responsive PD with prominent dysphasia, dysarthria......, and cognitive decline. Longitudinal clinical follow-up, EEG, SPECT and CSF biomarker examinations suggested an underlying encephalopathy with cortical involvement. The mutated allele (c.209A) was present within a haplotype different from that shared among mutation carriers in the Italian (Contursi...

  10. Joint analysis of the NACP-REP1 marker within the alpha synuclein gene concludes association with alcohol dependence.

    Science.gov (United States)

    Bönsch, D; Lederer, T; Reulbach, U; Hothorn, T; Kornhuber, J; Bleich, S

    2005-04-01

    Various studies have linked alcohol dependence phenotypes to chromosome 4. One candidate gene is NACP (non-amyloid component of plaques), coding for alpha synuclein. Recently, it has been shown that alpha synuclein mRNA is increased in alcohol-dependent patients within withdrawal state. This increase is significantly associated with craving, especially obsessive craving. On the basis of these observations, the present study analysed two polymorphic repeats within the NACP gene. We found highly significant longer alleles of NACP-REP1 in alcohol-dependent patients compared with healthy controls (Kruskal-Wallis test, chi(2)=99.5; df=3, Pcraving, a key factor in the genesis and maintenance not only of alcoholism but also of addiction in general.

  11. Differential effects of UCHL1 modulation on alpha-synuclein in PD-like models of alpha-synucleinopathy.

    Directory of Open Access Journals (Sweden)

    Anna E Cartier

    Full Text Available Parkinson's disease (PD is a progressive neurodegenerative disorder caused by genetic and environmental factors. Abnormal accumulation and aggregation of alpha-synuclein (a-syn within neurons, and mutations in the a-syn and UCH-L1 genes have been shown to play a role in the pathogenesis of PD. In light of recent reports suggesting an interaction between a-synuclein and UCH-L1, we investigated the effects of UCH-L1 inhibition on a-syn distribution and expression levels in primary neurons and hippocampal tissues derived from non transgenic (non tg and a-syn over expressing tg mice. We show that suppression of UCH-L1 activity increased a-syn levels in control, non tg neurons, and resulted in a concomitant accumulation of presynaptic a-syn in these neurons. In contrast, blocking UCH-L1 activity in a-syn over expressing neurons decreased a-syn levels, and enhanced its synaptic clearance. In vitro studies verified the LDN-induced inhibition of UCH-L1 had minimal effect on LC3 (a marker of autophagy in control cells, in cells over expressing a-syn UCH-L1 inhibition resulted in increased LC3 activity. These findings suggest a possible differential role of UCH-L1 function under normal and pathological conditions. Furthermore, in the context of a-syn-induced pathology, modulation of UCH-L1 activity could serve as a therapeutic tool to enhance the autophagy pathway and induce clearance of the observed accumulated/aggregated a-syn species in the PD brain.

  12. Protective effect of alpha-synuclein knockdown on methamphetamine-induced neurotoxicity in dopaminergic neurons

    Institute of Scientific and Technical Information of China (English)

    Yunchun Tai; Ling Chen; Enping Huang; Chao Liu; Xingyi Yang; Pingming Qiu; Huijun Wang

    2014-01-01

    The over-expression of α-synuclein is a major factor in the death of dopaminergic neurons in a methamphetamine-induced model of Parkinson’s disease. In the present study, α-synuclein knockdown rats were created by injecting α-synuclein-shRNA lentivirus stereotaxically into the right striatum of experimental rats. At 2 weeks post-injection, the rats were injected intraper-itoneally with methamphetamine to establish the model of Parkinson’s disease. Expression ofα-synuclein mRNA and protein in the right striatum of the injected rats was significantly down-regulated. Food intake and body weight were greater in α-synuclein knockdown rats, and water intake and stereotyped behavior score were lower than in model rats. Striatal dopamine and tyrosine hydroxylase levels were significantly elevated in α-synuclein knockdown rats. Moreover, superoxide dismutase activity was greater in α-synuclein knockdown rat striatum, but the levels of reactive oxygen species, malondialdehyde, nitric oxide synthase and nitrogen monoxide were lower compared with model rats. We also found that α-synuclein knockdown inhibited metham-phetamine-induced neuronal apoptosis. These results suggest that α-synuclein has the capacity to reverse methamphetamine-induced apoptosis of dopaminergic neurons in the rat striatum by inhibiting oxidative stress and improving dopaminergic system function.

  13. Protective effect of alpha-synuclein knockdown on methamphetamine-induced neurotoxicity in dopaminergic neurons

    OpenAIRE

    Tai, Yunchun; Chen, Ling; Huang, Enping; Liu, Chao; Yang, Xingyi; Qiu, Pingming; Wang, Huijun

    2014-01-01

    The over-expression of α-synuclein is a major factor in the death of dopaminergic neurons in a methamphetamine-induced model of Parkinson's disease. In the present study, α-synuclein knockdown rats were created by injecting α-synuclein-shRNA lentivirus stereotaxically into the right striatum of experimental rats. At 2 weeks post-injection, the rats were injected intraperitoneally with methamphetamine to establish the model of Parkinson's disease. Expression of α-synuclein mRNA and protein in ...

  14. Increased intestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in early Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Christopher B Forsyth

    Full Text Available UNLABELLED: Parkinson's disease (PD is the second most common neurodegenerative disorder of aging. The pathological hallmark of PD is neuronal inclusions termed Lewy bodies whose main component is alpha-synuclein protein. The finding of these Lewy bodies in the intestinal enteric nerves led to the hypothesis that the intestine might be an early site of PD disease in response to an environmental toxin or pathogen. One potential mechanism for environmental toxin(s and proinflammatory luminal products to gain access to mucosal neuronal tissue and promote oxidative stress is compromised intestinal barrier integrity. However, the role of intestinal permeability in PD has never been tested. We hypothesized that PD subjects might exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine. To test our hypothesis we evaluated intestinal permeability in subjects newly diagnosed with PD and compared their values to healthy subjects. In addition, we obtained intestinal biopsies from both groups and used immunohistochemistry to assess bacterial translocation, nitrotyrosine (oxidative stress, and alpha-synuclein. We also evaluated serum markers of endotoxin exposure including LPS binding protein (LBP. Our data show that our PD subjects exhibit significantly greater intestinal permeability (gut leakiness than controls. In addition, this intestinal hyperpermeability significantly correlated with increased intestinal mucosa staining for E. coli bacteria, nitrotyrosine, and alpha-synuclein as well as serum LBP levels in PD subjects. These data represent not only the first demonstration of abnormal intestinal permeability in PD subjects but also the first correlation of increased intestinal permeability in PD with intestinal alpha-synuclein (the hallmark of PD, as well as staining for gram negative bacteria and tissue oxidative stress. Our study may thus shed new light on PD pathogenesis as well as provide a new method for

  15. Reciprocal signals between microglia and neurons regulate alpha-synuclein secretion by exophagy through a neuronal cJU-N-Nterminal kinase-signaling axis

    DEFF Research Database (Denmark)

    Christensen, Dan Ploug; Ejlerskov, Patrick; Rasmussen, Izabela

    2016-01-01

    Background: Secretion of proteopathic alpha-synuclein (alpha-SNC) species from neurons is a suspected driving force in the propagation of Parkinson's disease (PD). We have previously implicated exophagy, the exocytosis of autophagosomes, as a dominant mechanism of alpha-SNC secretion in different...

  16. Concentration dependence of alpha-synuclein fibril length assessed by quantitative atomic force microscopy and statistical-mechanical theory.

    Science.gov (United States)

    van Raaij, Martijn E; van Gestel, Jeroen; Segers-Nolten, Ine M J; de Leeuw, Simon W; Subramaniam, Vinod

    2008-11-15

    The initial concentration of monomeric amyloidogenic proteins is a crucial factor in the in vitro formation of amyloid fibrils. We use quantitative atomic force microscopy to study the effect of the initial concentration of human alpha-synuclein on the mean length of mature alpha-synuclein fibrils, which are associated with Parkinson's disease. We determine that the critical initial concentration, below which low-molecular-weight species dominate and above which fibrils are the dominant species, lies at approximately 15 muM, in good agreement with earlier measurements using biochemical methods. In the concentration regime where fibrils dominate, we find that their mean length increases with initial concentration. These results correspond well to the qualitative predictions of a recent statistical-mechanical model of amyloid fibril formation. In addition, good quantitative agreement of the statistical-mechanical model with the measured mean fibril length as a function of initial protein concentration, as well as with the fibril length distributions for several protein concentrations, is found for reasonable values of the relevant model parameters. The comparison between theory and experiment yields, for the first time to our knowledge, an estimate of the magnitude of the free energies associated with the intermolecular interactions that govern alpha-synuclein fibril formation.

  17. Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo.

    Directory of Open Access Journals (Sweden)

    Elodie Angot

    Full Text Available Several people with Parkinson's disease have been treated with intrastriatal grafts of fetal dopaminergic neurons. Following autopsy, 10-22 years after surgery, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. Numerous studies have attempted to explain these findings in cell and animal models. In cell culture, α-synuclein has been found to transfer from one cell to another, via mechanisms that include exosomal transport and endocytosis, and in certain cases seed aggregation in the recipient cell. In animal models, transfer of α-synuclein from host brain cells to grafted neurons has been shown, but the reported frequency of the event has been relatively low and little is known about the underlying mechanisms as well as the fate of the transferred α-synuclein. We now demonstrate frequent transfer of α-synuclein from a rat brain engineered to overexpress human α-synuclein to grafted dopaminergic neurons. Further, we show that this model can be used to explore mechanisms underlying cell-to-cell transfer of α-synuclein. Thus, we present evidence both for the involvement of endocytosis in α-synuclein uptake in vivo, and for seeding of aggregation of endogenous α-synuclein in the recipient neuron by the transferred α-synuclein. Finally, we show that, at least in a subset of the studied cells, the transmitted α-synuclein is sensitive to proteinase K. Our new model system could be used to test compounds that inhibit cell-to-cell transfer of α-synuclein and therefore might retard progression of Parkinson neuropathology.

  18. Differences in extinction of conditioned fear in C57BL/6 substrains are unrelated to expression of alpha-synuclein.

    Science.gov (United States)

    Siegmund, Anja; Langnaese, Kristina; Wotjak, Carsten T

    2005-02-28

    C57BL/6 mice are commonly used as background strains for genetically modified mice, and little attention is usually paid to the notification of the specific substrain. However, it is known that C57BL/6NCrl (B6N) and C57BL/6JOlaHsd (B6JOla) mice differ in the course of extinction of conditioned fear (Stiedl O, Radulovic J, Lohmann R, Birkenfeld K, Palve M, Kammermeier J, et al. Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice. Behav Brain Res 1999;104:1-12), as well as in the expression of alpha-synuclein (Specht CG, Schoepfer R. Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci 2001;2:11). We tested for a causal relationship between the two findings by employing B6N (expressing alpha-synuclein), B6JOla (not expressing alpha-syn) and the third strain C57BL/6JCrl (B6Jax, expressing alpha-syn). We show that alpha-syn does not account for differences in extinction in a fear conditioning task, as its expression did not covary with the decrease of freezing on repeated non-reinforced tone and context exposure in the three strains: B6Jax exhibited fastest extinction followed by B6JOla. In contrast, B6N showed persistent fear over the course of extinction training. The differences in extinction between B6JOla and B6N were unrelated to sensorimotor processing (pain threshold and basal tone reaction) and innate fear (light-dark test). However, B6Jax displayed less innate fear than B6JOla and B6N. Our results of marked differences in innate and conditioned fear in three B6 substrains illustrate the necessity of a strict adherence to an exact mouse strain nomenclature.

  19. Exogenous Alpha-Synuclein Alters Pre- and Post-Synaptic Activity by Fragmenting Lipid Rafts.

    Science.gov (United States)

    Emanuele, Marco; Esposito, Alessandro; Camerini, Serena; Antonucci, Flavia; Ferrara, Silvia; Seghezza, Silvia; Catelani, Tiziano; Crescenzi, Marco; Marotta, Roberto; Canale, Claudio; Matteoli, Michela; Menna, Elisabetta; Chieregatti, Evelina

    2016-05-01

    Alpha-synuclein (αSyn) interferes with multiple steps of synaptic activity at pre-and post-synaptic terminals, however the mechanism/s by which αSyn alters neurotransmitter release and synaptic potentiation is unclear. By atomic force microscopy we show that human αSyn, when incubated with reconstituted membrane bilayer, induces lipid rafts' fragmentation. As a consequence, ion channels and receptors are displaced from lipid rafts with consequent changes in their activity. The enhanced calcium entry leads to acute mobilization of synaptic vesicles, and exhaustion of neurotransmission at later stages. At the post-synaptic terminal, an acute increase in glutamatergic transmission, with increased density of PSD-95 puncta, is followed by disruption of the interaction between N-methyl-d-aspartate receptor (NMDAR) and PSD-95 with ensuing decrease of long term potentiation. While cholesterol loading prevents the acute effect of αSyn at the presynapse; inhibition of casein kinase 2, which appears activated by reduction of cholesterol, restores the correct localization and clustering of NMDARs.

  20. Mitochondrial Dysfunction: The Road to Alpha-Synuclein Oligomerization in PD

    Directory of Open Access Journals (Sweden)

    A. R. Esteves

    2011-01-01

    Full Text Available While the etiology of Parkinson's disease remains largely elusive, there is accumulating evidence suggesting that mitochondrial dysfunction occurs prior to the onset of symptoms in Parkinson's disease. Mitochondria are remarkably primed to play a vital role in neuronal cell survival since they are key regulators of energy metabolism (as ATP producers, of intracellular calcium homeostasis, of NAD+/NADH ratio, and of endogenous reactive oxygen species production and programmed cell death. In this paper, we focus on mitochondrial dysfunction-mediated alpha-synuclein aggregation. We highlight some of the findings that provide proof of evidence for a mitochondrial metabolism control in Parkinson's disease, namely, mitochondrial regulation of microtubule-dependent cellular traffic and autophagic lysosomal pathway. The knowledge that microtubule alterations may lead to autophagic deficiency and may compromise the cellular degradation mechanisms that culminate in the progressive accumulation of aberrant protein aggregates shields new insights to the way we address Parkinson's disease. In line with this knowledge, an innovative window for new therapeutic strategies aimed to restore microtubule network may be unlocked.

  1. Alpha synuclein is transported into and out of the brain by the blood-brain barrier.

    Science.gov (United States)

    Sui, Yu-Ting; Bullock, Kristin M; Erickson, Michelle A; Zhang, Jing; Banks, W A

    2014-12-01

    Alpha-synuclein (α-Syn), a small protein with multiple physiological and pathological functions, is one of the dominant proteins found in Lewy Bodies, a pathological hallmark of Lewy body disorders, including Parkinson's disease (PD). More recently, α-Syn has been found in body fluids, including blood and cerebrospinal fluid, and is likely produced by both peripheral tissues and the central nervous system. Exchange of α-Syn between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications. However, little is known about the ability of α-Syn to cross the blood-brain barrier (BBB). Here, we found that radioactively labeled α-Syn crossed the BBB in both the brain-to-blood and the blood-to-brain directions at rates consistent with saturable mechanisms. Low-density lipoprotein receptor-related protein-1 (LRP-1), but not p-glycoprotein, may be involved in α-Syn efflux and lipopolysaccharide (LPS)-induced inflammation could increase α-Syn uptake by the brain by disrupting the BBB.

  2. Network Analysis Implicates Alpha-Synuclein (Snca) in the Regulation of Ovariectomy-Induced Bone Loss

    Science.gov (United States)

    Calabrese, Gina; Mesner, Larry D.; Foley, Patricia L.; Rosen, Clifford J.; Farber, Charles R.

    2016-01-01

    The postmenopausal period in women is associated with decreased circulating estrogen levels, which accelerate bone loss and increase the risk of fracture. Here, we gained novel insight into the molecular mechanisms mediating bone loss in ovariectomized (OVX) mice, a model of human menopause, using co-expression network analysis. Specifically, we generated a co-expression network consisting of 53 gene modules using expression profiles from intact and OVX mice from a panel of inbred strains. The expression of four modules was altered by OVX, including module 23 whose expression was decreased by OVX across all strains. Module 23 was enriched for genes involved in the response to oxidative stress, a process known to be involved in OVX-induced bone loss. Additionally, module 23 homologs were co-expressed in human bone marrow. Alpha synuclein (Snca) was one of the most highly connected “hub” genes in module 23. We characterized mice deficient in Snca and observed a 40% reduction in OVX-induced bone loss. Furthermore, protection was associated with the altered expression of specific network modules, including module 23. In summary, the results of this study suggest that Snca regulates bone network homeostasis and ovariectomy-induced bone loss. PMID:27378017

  3. Exogenous Alpha-Synuclein Alters Pre- and Post-Synaptic Activity by Fragmenting Lipid Rafts

    Directory of Open Access Journals (Sweden)

    Marco Emanuele

    2016-05-01

    Full Text Available Alpha-synuclein (αSyn interferes with multiple steps of synaptic activity at pre-and post-synaptic terminals, however the mechanism/s by which αSyn alters neurotransmitter release and synaptic potentiation is unclear. By atomic force microscopy we show that human αSyn, when incubated with reconstituted membrane bilayer, induces lipid rafts' fragmentation. As a consequence, ion channels and receptors are displaced from lipid rafts with consequent changes in their activity. The enhanced calcium entry leads to acute mobilization of synaptic vesicles, and exhaustion of neurotransmission at later stages. At the post-synaptic terminal, an acute increase in glutamatergic transmission, with increased density of PSD-95 puncta, is followed by disruption of the interaction between N-methyl-d-aspartate receptor (NMDAR and PSD-95 with ensuing decrease of long term potentiation. While cholesterol loading prevents the acute effect of αSyn at the presynapse; inhibition of casein kinase 2, which appears activated by reduction of cholesterol, restores the correct localization and clustering of NMDARs.

  4. High-Throughput Screening Methodology to Identify Alpha-Synuclein Aggregation Inhibitors.

    Science.gov (United States)

    Pujols, Jordi; Peña-Díaz, Samuel; Conde-Giménez, María; Pinheiro, Francisca; Navarro, Susanna; Sancho, Javier; Ventura, Salvador

    2017-03-02

    An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson's disease, this process involves the aggregation of alpha-synuclein (α-syn) into intraneuronal inclusions. Thus, compounds that inhibit α-syn aggregation represent a promising therapeutic strategy as disease-modifying agents for neurodegeneration. The formation of α-syn amyloid aggregates can be reproduced in vitro by incubation of the recombinant protein. However, the in vitro aggregation of α-syn is exceedingly slow and highly irreproducible, therefore precluding fast high throughput anti-aggregation drug screening. Here, we present a simple and easy-to-implement in-plate method for screening large chemical libraries in the search for α-syn aggregation modulators. It allows us to monitor aggregation kinetics with high reproducibility, while being faster and requiring lower protein amounts than conventional aggregation assays. We illustrate how the approach enables the identification of strong aggregation inhibitors in a library of more than 14,000 compounds.

  5. Role of alpha-synuclein in neuronal apoptosis induced by rotenone

    Institute of Scientific and Technical Information of China (English)

    Yanying Liu; Hui Yang

    2006-01-01

    BACKGROUND: Aggregation of α-synuclein is the major component of Lewy bodies, which are the pathological hallmarks of Parkinson disease (PD). Although the mechanism of this protein aggregates is unclear,previous study showed that environmental toxins such as rotenone could induce the expression and aggregation of α-synuclein.OBJECTIVE: To observe the role of α-synuclein in PD.DESIGN: A randomized controlled trial.SETTING: Beijing Institute for Neuroscience, Capital University of Medical Sciences.MATERIALS: This study was performed from July 2005 to January 2006 at the Beijing Institute for Neuroscience, Capital University of Medical Sciences. Human dopaminergic neuroblastoma SH-SY5Y cells were provided by Beijing Institute for Neuroscience, Capital University of Medical Sciences.METHODS: Human dopaminergic neuroblastoma SH-SY5Y cells were treated to make α-synuclein over express. Rotenone was added into the medium of cultured both native SH-SY5Y cells and α-synuclein-overexpression SH-SY5Y cells. Lactate dehydrogenase (LDH) assay was used to detect with the cell viability. Flow cytometry and electrophoresis were adopted to measure the cell apoptosis.MAIN OUTCOME MEASURES: Cell viability, DNA fragmentation, and the number of cell apoptosis.RESULTS: After being treated with rotenone, LDH activity of α-synuclein overexpressed SH-SY5Y cells was (76.625±6.34) μ kat/L, which was significantly lower than that of control group (P < 0.05). As compared with normal SH-SY5Y cell, α-synuclein over-expressed SH-SY5Y cells had less DNA fragments and apoptotic cells. Α-synuclein might play a role in cell apoptosis induced by rotenone, which was also confirmed by using of antioxidant reagent.CONCLUSION: α-synuclein may partially protect against cell apoptosis induced by rotenone in SH-SY5Y cells.

  6. Alpha-Synuclein Stimulation of Astrocytes: Potential Role for Neuroinflammation and Neuroprotection

    Directory of Open Access Journals (Sweden)

    He-Jin Lee

    2010-01-01

    Full Text Available Selective loss of neurons, abnormal protein deposition and neuroinflammation are the common pathological features of neurodegenerative diseases, and these features are closely related to one another. In Parkinson's disease, abnormal aggregation and deposition of α-synuclein is known as a critical event in pathogenesis of the disease, as well as in other related neurodegenerative disorders, such as dementia with Lewy bodies and multiple system atrophy. Increasing evidence suggests that α-synuclein aggregates can activate glial cells to induce neuroinflammation. However, how an inflammatory microenvironment is established and maintained by this protein remains unknown. Findings from our recent study suggest that neuronal α-synuclein can be directly transferred to astrocytes through sequential exocytosis and endocytosis and induce inflammatory responses from astrocytes. Here we discuss potential roles of astrocytes in a cascade of events leading to α-synuclein-induced neuroinflammation.

  7. Alpha-synuclein oligomers - neurotoxic molecules in Parkinson’s disease and other Lewy body disorders

    Directory of Open Access Journals (Sweden)

    Martin Ingelsson

    2016-09-01

    Full Text Available Adverse intra- and extracellular effects of toxic α-synuclein are believed to be central to the pathogenesis in Parkinson’s disease and other disorders with Lewy body pathology in the nervous system. One of the physiological roles of α-synuclein relates to the regulation of neurotransmitter release at the presynapse, although it is still unclear whether this mechanism depends on the action of monomers or smaller oligomers. As for the pathogenicity, accumulating evidence suggest that prefibrillar species, rather than the deposits per se, are responsible for the toxicity in affected cells. In particular, larger oligomers or protofibrils of α-synuclein have been shown to impair protein degradation as well as the function of several organelles, such as the mitochondria and the endoplasmic reticulum. Accumulating evidence further suggest that oligomers/protofibrils may have a toxic effect on the synapse, which may lead to disrupted electrophysiological properties. In addition, recent data indicate that oligomeric α-synuclein species can spread between cells, either as free-floating proteins or via extracellular vesicles, and thereby act as seeds to propagate disease between interconnected brain regions. Taken together, several lines of evidence suggest that α-synuclein have neurotoxic properties and therefore should be an appropriate molecular target for therapeutic intervention in Parkinson’s disease and other disorders with Lewy pathology. In this context, immunotherapy with monoclonal antibodies against α-synuclein oligomers/protofibrils should be a particularly attractive treatment option.

  8. Alpha-synuclein pathology and axonal degeneration of the peripheral motor nerves innervating pharyngeal muscles in Parkinson disease.

    Science.gov (United States)

    Mu, Liancai; Sobotka, Stanislaw; Chen, Jingming; Su, Hungxi; Sanders, Ira; Adler, Charles H; Shill, Holly A; Caviness, John N; Samanta, Johan E; Beach, Thomas G

    2013-02-01

    Parkinson disease (PD) is a neurodegenerative disease primarily characterized by cardinal motor manifestations and CNS pathology. Current drug therapies can often stabilize these cardinal motor symptoms, and attention has shifted to the other motor and nonmotor symptoms of PD that are resistant to drug therapy. Dysphagia in PD is perhaps the most important drug-resistant symptom because it leads to aspiration and pneumonia, the leading cause of death. Here, we present direct evidence for degeneration of the pharyngeal motor nerves in PD. We examined the cervical vagal nerve (cranial nerve X), pharyngeal branch of nerve X, and pharyngeal plexus innervating the pharyngeal muscles in 14 postmortem specimens, that is, from 10 patients with PD and 4 age-matched control subjects. Synucleinopathy in the pharyngeal nerves was detected using an immunohistochemical method for phosphorylated α-synuclein. Alpha-synuclein aggregates were revealed in nerve X and the pharyngeal branch of nerve X, and immunoreactive intramuscular nerve twigs and axon terminals within the neuromuscular junctions were identified in all of the PD patients but in none of the controls. These findings indicate that the motor nervous system of the pharynx is involved in the pathologic process of PD. Notably, PD patients who have had dysphagia had a higher density of α-synuclein aggregates in the pharyngeal nerves than those without dysphagia. These findings indicate that motor involvement of the pharynx in PD is one of the factors leading to oropharyngeal dysphagia commonly seen in PD patients.

  9. Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation.

    Directory of Open Access Journals (Sweden)

    Diana F Lázaro

    2014-11-01

    Full Text Available Aggregation of alpha-synuclein (ASYN in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

  10. Collaborative analysis of alpha-synuclein gene promoter variability and Parkinson disease.

    Science.gov (United States)

    Maraganore, Demetrius M; de Andrade, Mariza; Elbaz, Alexis; Farrer, Matthew J; Ioannidis, John P; Krüger, Rejko; Rocca, Walter A; Schneider, Nicole K; Lesnick, Timothy G; Lincoln, Sarah J; Hulihan, Mary M; Aasly, Jan O; Ashizawa, Tetsuo; Chartier-Harlin, Marie-Christine; Checkoway, Harvey; Ferrarese, Carlo; Hadjigeorgiou, Georgios; Hattori, Nobutaka; Kawakami, Hideshi; Lambert, Jean-Charles; Lynch, Timothy; Mellick, George D; Papapetropoulos, Spiridon; Parsian, Abbas; Quattrone, Aldo; Riess, Olaf; Tan, Eng-King; Van Broeckhoven, Christine

    2006-08-09

    Identification and replication of susceptibility genes for Parkinson disease at the population level have been hampered by small studies with potential biases. Alpha-synuclein (SNCA) has been one of the most promising susceptibility genes, but large-scale studies have been lacking. To determine whether allele-length variability in the dinucleotide repeat sequence (REP1) of the SNCA gene promoter is associated with Parkinson disease susceptibility, whether SNCA promoter haplotypes are associated with Parkinson disease, and whether REP1 variability modifies age at onset. We performed a collaborative analysis of individual-level data on SNCA REP1 and flanking markers in patients with Parkinson disease and controls. Study site recruitment, data collection, and analyses were performed between April 5, 2004, and December 31, 2005. Eighteen participating sites of a global genetics consortium provided clinical data. Genotyping was performed for SNCA REP1, -770, and -116 markers at individual sites; however, each site also provided 20 DNA samples for regenotyping centrally. Measures included estimations of Hardy-Weinberg equilibrium in controls; a test of heterogeneity; analyses for association of single variants or haplotypes; and survival analyses for age at onset. Of the 18 sites, 11 met stringent criteria for concordance with Hardy-Weinberg equilibrium and low genotyping error rate. These 11 sites provided complete data for 2692 cases and 2652 controls. There was no heterogeneity across studies (P>.60). The SNCA REP1 alleles differed in frequency for cases and controls (PParkinson disease (odds ratio, 1.43; 95% confidence interval, 1.22-1.69; PParkinson disease only when they included REP1 as one of the loci. However, genotypes defined by REP1 alleles did not modify age at onset (P = .55). This large-scale collaborative analysis demonstrates that SNCA REP1 allele-length variability is associated with an increased risk of Parkinson disease.

  11. Trehalose does not improve neuronal survival on exposure to alpha-synuclein pre-formed fibrils

    Directory of Open Access Journals (Sweden)

    Matthew Redmann

    2017-04-01

    Full Text Available Parkinson's disease is a debilitating neurodegenerative disorder that is pathologically characterized by intracellular inclusions comprised primarily of alpha-synuclein (αSyn that can also be transmitted from neuron to neuron. Several lines of evidence suggest that these inclusions cause neurodegeneration. Thus exploring strategies to improve neuronal survival in neurons with αSyn aggregates is critical. Previously, exposure to αSyn pre-formed fibrils (PFFs has been shown to induce aggregation of endogenous αSyn resulting in cell death that is exacerbated by either starvation or inhibition of mTOR by rapamycin, both of which are able to induce autophagy, an intracellular protein degradation pathway. Since mTOR inhibition may also inhibit protein synthesis and starvation itself can be detrimental to neuronal survival, we investigated the effects of autophagy induction on neurons with αSyn inclusions by a starvation and mTOR-independent autophagy induction mechanism. We exposed mouse primary cortical neurons to PFFs to induce inclusion formation in the presence and absence of the disaccharide trehalose, which has been proposed to induce autophagy and stimulate lysosomal biogenesis. As expected, we observed that on exposure to PFFs, there was increased abundance of pS129-αSyn aggregates and cell death. Trehalose alone increased LC3-II levels, consistent with increased autophagosome levels that remained elevated with PFF exposure. Interestingly, trehalose alone increased cell viability over a 14-d time course. Trehalose was also able to restore cell viability to control levels, but PFFs still exhibited toxic effects on the cells. These data provide essential information regarding effects of trehalose on αSyn accumulation and neuronal survival on exposure to PFF.

  12. Modelling Ser129 phosphorylation inhibits membrane binding of pore-forming alpha-synuclein oligomers.

    Directory of Open Access Journals (Sweden)

    Georg Sebastian Nübling

    Full Text Available BACKGROUND: In several neurodegenerative diseases, hyperphosphorylation at position Ser129 is found in fibrillar deposits of alpha-synuclein (asyn, implying a pathophysiological role of asyn phosphorylation in neurodegeneration. However, recent animal models applying asyn phosphorylation mimics demonstrated a protective effect of phosphorylation. Since metal-ion induced asyn oligomers were identified as a potential neurotoxic aggregate species with membrane pore-forming abilities, the current study was undertaken to determine effects of asyn phosphorylation on oligomer membrane binding. METHODS: We investigated the influence of S129 phosphorylation on interactions of metal-ion induced asyn oligomers with small unilamellar lipid vesicles (SUV composed of POPC and DPPC applying the phosphorylation mimic asyn129E. Confocal single-particle fluorescence techniques were used to monitor membrane binding at the single-particle level. RESULTS: Binding of asyn129E monomers to gel-state membranes (DPPC-SUV is slightly reduced compared to wild-type asyn, while no interactions with membranes in the liquid-crystalline state (POPC-SUV are seen for both asyn and asyn129E. Conversely, metal-ion induced oligomer formation is markedly increased in asyn129E. Surprisingly, membrane binding to POPC-SUV is nearly absent in Fe(3+ induced asyn129E oligomers and markedly reduced in Al(3+ induced oligomers. CONCLUSION: The protective effect of pseudophosphorylation seen in animal models may be due to impeded oligomer membrane binding. Phosphorylation at Ser129 may thus have a protective effect against neurotoxic asyn oligomers by preventing oligomer membrane binding and disruption of the cellular electrophysiological equilibrium. Importantly, these findings put a new complexion on experimental pharmaceutical interventions against POLO-2 kinase.

  13. Is alpha-synuclein loss-of-function a contributor to parkinsonian pathology? Evidence from non-human primates

    Directory of Open Access Journals (Sweden)

    Timothy J Collier

    2016-01-01

    Full Text Available Accumulation of alpha-synuclein (α-syn in Lewy bodies and neurites of midbrain dopamine neurons is diagnostic for Parkinson’s disease (PD, leading to the proposal that PD is a toxic gain-of-function synucleinopathy. Here we discuss the alternative viewpoint that α-syn displacement from synapses by misfolding and aggregation results in a toxic loss-of-function. In support of this hypothesis we provide evidence from our pilot study demonstrating that knockdown of endogenous α-syn in dopamine neurons of nonhuman primates reproduces the pattern of nigrostriatal degeneration characteristic of PD.

  14. Direct and/or Indirect Roles for SUMO in Modulating Alpha-Synuclein Toxicity

    Directory of Open Access Journals (Sweden)

    Shamini Vijayakumaran

    2015-07-01

    Full Text Available α-Synuclein inclusion bodies are a pathological hallmark of several neurodegenerative diseases, including Parkinson’s disease, and contain aggregated α-synuclein and a variety of recruited factors, including protein chaperones, proteasome components, ubiquitin and the small ubiquitin-like modifier, SUMO-1. Cell culture and animal model studies suggest that misfolded, aggregated α-synuclein is actively translocated via the cytoskeletal system to a region of the cell where other factors that help to lessen the toxic effects can also be recruited. SUMO-1 covalently conjugates to various intracellular target proteins in a way analogous to ubiquitination to alter cellular distribution, function and metabolism and also plays an important role in a growing list of cellular pathways, including exosome secretion and apoptosis. Furthermore, SUMO-1 modified proteins have recently been linked to cell stress responses, such as oxidative stress response and heat shock response, with increased SUMOylation being neuroprotective in some cases. Several recent studies have linked SUMOylation to the ubiquitin-proteasome system, while other evidence implicates the lysosomal pathway. Other reports depict a direct mechanism whereby sumoylation reduced the aggregation tendency of α-synuclein, and reduced the toxicity. However, the precise role of SUMO-1 in neurodegeneration remains unclear. In this review, we explore the potential direct or indirect role(s of SUMO-1 in the cellular response to misfolded α-synuclein in neurodegenerative disorders.

  15. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

    Science.gov (United States)

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M; Zavala-Flores, Laura; Reyes-Reyes, Elsa M; Seravalli, Javier; Stanciu, Lia A; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2015-09-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

  16. The role of alpha-synuclein in melanin synthesis in melanoma and dopaminergic neuronal cells.

    Directory of Open Access Journals (Sweden)

    Tianhong Pan

    Full Text Available The relatively high co-occurrence of Parkinson's disease (PD and melanoma has been established by a large number of epidemiological studies. However, a clear biological explanation for this finding is still lacking. Ultra-violet radiation (UVR-induced skin melanin synthesis is a defense mechanism against UVR-induced damage relevant to the initiation of melanoma, whereas, increased neuromelanin (NM, the melanin synthesized in dopaminergic neurons, may enhance the susceptibility to oxidative stress-induced neuronal injury relevant to PD. SNCA is a PD-causing gene coding for alpha-Synuclein (α-Syn that expresses not only in brain, but also in skin as well as in tumors, such as melanoma. The findings that α-Syn can interact with tyrosinase (TYR and inhibit tyrosine hydroxylase (TH, both of which are enzymes involved in the biosynthesis of melanin and dopamine (DA, led us to propose that α-Syn may participate in the regulation of melanin synthesis. In this study, by applying ultraviolet B (UVB light, a physiologically relevant stimulus of melanogenesis, we detected melanin synthesis in A375 and SK-MEL-28 melanoma cells and in SH-SY5Y and PC12 dopaminergic neuronal cells and determined effects of α-Syn on melanin synthesis. Our results showed that UVB light exposure increased melanin synthesis in all 4 cell lines. However, we found that α-Syn expression reduced UVB light-induced increase of melanin synthesis and that melanin content was lower when melanoma cells were expressed with α-Syn, indicating that α-Syn may have inhibitory effects on melanin synthesis in melanoma cells. Different from melanoma cells, the melanin content was higher in α-Syn-over-expressed dopaminergic neuronal SH-SY5Y and PC12 cells, cellular models of PD, than that in non-α-Syn-expressed control cells. We concluded that α-Syn could be one of the points responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in

  17. Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Sandra Tenreiro

    2014-05-01

    Full Text Available Alpha-synuclein (aSyn is the main component of proteinaceous inclusions known as Lewy bodies (LBs, the typical pathological hallmark of Parkinson's disease (PD and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼ 90% of aSyn in LBs is phosphorylated at S129 (pS129. Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.

  18. Long-term polarization of microglia upon alpha-synuclein overexpression in nonhuman primates

    DEFF Research Database (Denmark)

    Barkholt, Pernille; Sanchez-Guajardo, Vanesa Maria; Kirik, Denis

    2012-01-01

    We have previously shown that persistent ﰇ-sy- nuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) ﰇ-synuclein....

  19. Seeded aggregation and toxicity of {alpha}-synuclein and tau: cellular models of neurodegenerative diseases.

    Science.gov (United States)

    Nonaka, Takashi; Watanabe, Sayuri T; Iwatsubo, Takeshi; Hasegawa, Masato

    2010-11-05

    The deposition of amyloid-like filaments in the brain is the central event in the pathogenesis of neurodegenerative diseases. Here we report cellular models of intracytoplasmic inclusions of α-synuclein, generated by introducing nucleation seeds into SH-SY5Y cells with a transfection reagent. Upon introduction of preformed seeds into cells overexpressing α-synuclein, abundant, highly filamentous α-synuclein-positive inclusions, which are extensively phosphorylated and ubiquitinated and partially thioflavin-positive, were formed within the cells. SH-SY5Y cells that formed such inclusions underwent cell death, which was blocked by small molecular compounds that inhibit β-sheet formation. Similar seed-dependent aggregation was observed in cells expressing four-repeat Tau by introducing four-repeat Tau fibrils but not three-repeat Tau fibrils or α-synuclein fibrils. No aggregate formation was observed in cells overexpressing three-repeat Tau upon treatment with four-repeat Tau fibrils. Our cellular models thus provide evidence of nucleation-dependent and protein-specific polymerization of intracellular amyloid-like proteins in cultured cells.

  20. Tri- and pentamethine cyanine dyes for fluorescent detection of alpha-synuclein oligomeric aggregates

    NARCIS (Netherlands)

    Kovalska, V.B.; Yu Losytskyy, M.; Tolmachev, O.I.; Slominskii, Yu.L.; Segers-Nolten, G.M.J.; Subramaniam, V.; Yarmoluk, S.M.

    2012-01-01

    The pathogenesis of Parkinson’s disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of α-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligomeri

  1. Explorations of the application of cyanine dyes for quantitative alpha-synuclein detection

    NARCIS (Netherlands)

    Volkova, K.D.; Kovalska, V.B.; Segers-Nolten, Gezina M.J.; Veldhuis, G.; Veldhuis, G.J.; Subramaniam, Vinod; Yarmoluk, S.M.

    2009-01-01

    We examined the practical aspects of using fluorescent mono (T-284) and trimethinecyanine (SH-516) dyes for detecting and quantifying fibrillar α-synuclein (ASN). We studied the interaction of cyanine dyes with fibrillar proteins using fluorescence spectroscopy and atomic force microscopy. The comme

  2. Membrane-Bound Alpha Synuclein Clusters Induce Impaired Lipid Diffusion and Increased Lipid Packing.

    NARCIS (Netherlands)

    Iyer, Aditya; Schilderink, Nathalie; Claessens, Mireille M A E; Subramaniam, Vinod

    2016-01-01

    The aggregation of membrane-bound α-synuclein (αS) into oligomers and/or amyloid fibrils has been suggested to cause membrane damage in in vitro model phospholipid membrane systems and in vivo. In this study, we investigate how αS interactions that precede the formation of well-defined aggregates in

  3. Impairment of mitochondria dynamics by human A53T alpha-synuclein and rescue by NAP (davunetide) in a cell model for Parkinson's disease

    NARCIS (Netherlands)

    Q. Melo, T.; van Zomeren, K. C.; Ferrari, M F R; Boddeke, H. W. G. M.; Copray, J. C. V. M.

    The formation of oligomers and aggregates of overexpressed or mutant alpha-synuclein play a role in the degeneration of dopaminergic neurons in Parkinson's disease by causing dysfunction of mitochondria, reflected in their disturbed mobility and production of ROS. The mode of action and mechanisms

  4. Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy

    NARCIS (Netherlands)

    Roeters, Steven J.; Iyer, Aditya; Pletikapic, Galja; Kogan, Vladimir; Subramaniam, Vinod; Woutersen, Sander

    2017-01-01

    The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson’s disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fi

  5. Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways.

    Science.gov (United States)

    Almandoz-Gil, Leire; Welander, Hedvig; Ihse, Elisabet; Khoonsari, Payam Emami; Musunuri, Sravani; Lendel, Christofer; Sigvardson, Jessica; Karlsson, Mikael; Ingelsson, Martin; Kultima, Kim; Bergström, Joakim

    2017-09-01

    Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Elucidating the Role of Site-Specific Nitration of α-Synuclein in the Pathogenesis of Parkinson's Disease via Protein Semisynthesis and Mutagenesis.

    Science.gov (United States)

    Burai, Ritwik; Ait-Bouziad, Nadine; Chiki, Anass; Lashuel, Hilal A

    2015-04-22

    Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of intraneuronal inclusions consisting of aggregated and post-translationally modified α-synuclein (α-syn). Despite advances in the chemical synthesis of α-syn and other proteins, the generation of site-specifically nitrated synthetic proteins has not been reported. Consequently, it has not been possible to determine the roles of nitration at specific residues in regulating the physiological and pathogenic properties of α-syn. Here we report, for the first time, the site-specific incorporation of 3-nitrotyrosine at different regions of α-syn using native chemical ligation combined with a novel desulfurization strategy. This strategy enabled us to investigate the role of nitration at single or multiple tyrosine residues in regulating α-syn structure, membrane binding, oligomerization, and fibrils formation. We demonstrate that different site-specifically nitrated α-syn species exhibit distinct structural and aggregation properties and exhibit reduced affinity to negatively charged vesicle membranes. We provide evidence that intermolecular interactions between the N- and C-terminal regions of α-syn play critical roles in mediating nitration-induced α-syn oligomerization. For example, when Y39 is not available for nitration (Y39F and Y39/125F), the extent of cross-linking is limited mostly to dimer formation, whereas mutants in which Y39 along with one or multiple C-terminal tyrosines (Y125F, Y133F, Y136F and Y133/136F) can still undergo nitration readily to form higher-order oligomers. Our semisynthetic strategy for generating site-specifically nitrated proteins opens up new possibilities for investigating the role of nitration in regulating protein structure and function in health and disease.

  7. Inhibition of prolyl oligopeptidase increases the survival of alpha-synuclein overexpressing cells after rotenone exposure by reducing alpha-synuclein oligomers.

    Science.gov (United States)

    Dokleja, Lana; Hannula, Mirva J; Myöhänen, Timo T

    2014-11-07

    α-Synuclein (aSyn) aggregation, mitochondrial dysfunction and oxidative damage has been shown to be related to the death of dopaminergic neurons in Parkinson's disease (PD). Prolyl oligopeptidase (PREP) is proposed to increase aSyn aggregation, and PREP inhibition has been shown to inhibit the aggregation process in vitro and in vivo. In this study, we investigated the effects of a specific PREP inhibitor, KYP-2047, on rotenone induced aSyn aggregation and increased the production of reactive oxygen species (ROS) in cells overexpressing A53T mutation of aSyn. Rotenone, a mitochondrial toxin that induces oxidative damage and aSyn aggregation, associated with PD pathology, was selected as a model for this study. The results showed that rotenone induced the formation of high-molecular-weight aSyn oligomers, and this was countered by simultaneous incubation with KYP-2047. Inhibition of PREP also decreased the production of ROS in [A53T]aSyn overexpressing cells, leading to improved cell viability.

  8. Physiological and Pathological Role of Alpha-Synuclein in Parkinson’s Disease through Iron Mediated Oxidative Stress; The Role of a Putative Iron-Responsive Element

    OpenAIRE

    David Olivares; Xudong Huang; Lars Branden; Greig, Nigel H.; Jack T. Rogers

    2009-01-01

    Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder after Alzheimer’s disease (AD) and represents a large health burden to society. Genetic and oxidative risk factors have been proposed as possible causes, but their relative contribution remains unclear. Dysfunction of alpha-synuclein (\\(\\alpha\\)-syn) has been associated with PD due to its increased presence, together with iron, in Lewy bodies. Brain oxidative damage caused by iron may be partly mediated ...

  9. Effects of Trehalose on Thermodynamic Properties of Alpha-synuclein Revealed through Synchrotron Radiation Circular Dichroism

    Science.gov (United States)

    Ruzza, Paolo; Hussain, Rohanah; Biondi, Barbara; Calderan, Andrea; Tessari, Isabella; Bubacco, Luigi; Siligardi, Giuliano

    2015-01-01

    Many neurodegenerative diseases, including Huntington’s, Alzheimer’s and Parkinson’s diseases, are characterized by protein misfolding and aggregation. The capability of trehalose to interfere with protein misfolding and aggregation has been recently evaluated by several research groups. In the present work, we studied, by means of synchrotron radiation circular dichroism (SRCD) spectroscopy, the dose-effect of trehalose on α-synuclein conformation and/or stability to probe the capability of this osmolyte to interfere with α-synuclein’s aggregation. Our study indicated that a low trehalose concentration stabilized α-synuclein folding much better than at high concentration by blocking in vitro α-synuclein’s polymerisation. These results suggested that trehalose could be associated with other drugs leading to a new approach for treating Parkinson’s and other brain-related diseases. PMID:25946077

  10. Effects of Trehalose on Thermodynamic Properties of Alpha-synuclein Revealed through Synchrotron Radiation Circular Dichroism

    Directory of Open Access Journals (Sweden)

    Paolo Ruzza

    2015-05-01

    Full Text Available Many neurodegenerative diseases, including Huntington’s, Alzheimer’s and Parkinson’s diseases, are characterized by protein misfolding and aggregation. The capability of trehalose to interfere with protein misfolding and aggregation has been recently evaluated by several research groups. In the present work, we studied, by means of synchrotron radiation circular dichroism (SRCD spectroscopy, the dose-effect of trehalose on α-synuclein conformation and/or stability to probe the capability of this osmolyte to interfere with α-synuclein’s aggregation. Our study indicated that a low trehalose concentration stabilized α-synuclein folding much better than at high concentration by blocking in vitro α-synuclein’s polymerisation. These results suggested that trehalose could be associated with other drugs leading to a new approach for treating Parkinson’s and other brain-related diseases.

  11. Mutant alpha-synuclein causes age-dependent neuropathology in monkey brain.

    Science.gov (United States)

    Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua; Li, Xiao-Jiang

    2015-05-27

    Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2-3, 7-8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD.

  12. 5-HT2A Receptor Binding in the Frontal Cortex of Parkinson's Disease Patients and Alpha-Synuclein Overexpressing Mice

    DEFF Research Database (Denmark)

    Rasmussen, Nadja Bredo; Olesen, Mikkel Vestergaard; Brudek, Tomasz;

    2016-01-01

    function are also steadily being associated with this disease. Not much is known about the pathophysiology behind this. The aim of this study was thereby twofold: (1) to investigate receptor binding levels in Parkinson’s brains and (2) to investigate whether PD associated pathology, alpha-synuclein (AS...... by increased receptor binding in PD brains. In a separate study, we looked for changes in receptors in the prefrontal cortex in 52-week-old transgenic mice overexpressing human AS. We performed region-specific receptor binding measurements followed by gene expression analysis. The transgenic mice showed lower...... binding in the frontal association cortex that was not accompanied by changes in gene expression levels. This study is one of the first to look at differences in serotonin receptor levels in PD and in relation to AS overexpression....

  13. Alpha-synuclein, epigenetics, mitochondria, metabolism, calcium traffic, & circadian dysfunction in Parkinson's disease. An integrated strategy for management.

    Science.gov (United States)

    Phillipson, Oliver T

    2017-10-03

    The motor deficits which characterise the sporadic form of Parkinson's disease arise from age-related loss of a subset of dopamine neurons in the substantia nigra. Although motor symptoms respond to dopamine replacement therapies, the underlying disease process remains. This review details some features of the progressive molecular pathology and proposes deployment of a combination of nutrients: R-lipoic acid, acetyl-L-carnitine, ubiquinol, melatonin (or receptor agonists) and vitamin D3, with the collective potential to slow progression of these features. The main nutrient targets include impaired mitochondria and the associated oxidative/nitrosative stress, calcium stress and impaired gene transcription induced by pathogenic forms of alpha- synuclein. Benefits may be achieved via nutrient influence on epigenetic signaling pathways governing transcription factors for mitochondrial biogenesis, antioxidant defences and the autophagy-lysosomal pathway, via regulation of the metabolic energy sensor AMP activated protein kinase (AMPK) and the mammalian target of rapamycin mTOR. Nutrients also benefit expression of the transcription factor for neuronal survival (NR4A2), trophic factors GDNF and BDNF, and age-related calcium signals. In addition a number of non-motor related dysfunctions in circadian control, clock genes and associated metabolic, endocrine and sleep-wake activity are briefly addressed, as are late-stage complications in respect of cognitive decline and osteoporosis. Analysis of the network of nutrient effects reveals how beneficial synergies may counter the accumulation and promote clearance of pathogenic alpha-synuclein. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  14. Bioinorganic chemistry of Parkinson's disease: structural determinants for the copper-mediated amyloid formation of alpha-synuclein.

    Science.gov (United States)

    Binolfi, Andrés; Rodriguez, Esaú E; Valensin, Daniela; D'Amelio, Nicola; Ippoliti, Emiliano; Obal, Gonzalo; Duran, Rosario; Magistrato, Alessandra; Pritsch, Otto; Zweckstetter, Markus; Valensin, Gianni; Carloni, Paolo; Quintanar, Liliana; Griesinger, Christian; Fernández, Claudio O

    2010-11-15

    The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.

  15. In vivo visualization of alpha-synuclein deposition by carbon-11-labelled 2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy]benzoxazole positron emission tomography in multiple system atrophy.

    Science.gov (United States)

    Kikuchi, Akio; Takeda, Atsushi; Okamura, Nobuyuki; Tashiro, Manabu; Hasegawa, Takafumi; Furumoto, Shozo; Kobayashi, Michiko; Sugeno, Naoto; Baba, Toru; Miki, Yasuo; Mori, Fumiaki; Wakabayashi, Koichi; Funaki, Yoshihito; Iwata, Ren; Takahashi, Shoki; Fukuda, Hiroshi; Arai, Hiroyuki; Kudo, Yukitsuka; Yanai, Kazuhiko; Itoyama, Yasuto

    2010-06-01

    The histopathological hallmark of multiple system atrophy is the appearance of intracellular inclusion bodies, named glial cytoplasmic inclusions, which are mainly composed of alpha-synuclein fibrils. In vivo visualization of alpha-synuclein deposition should be used for the diagnosis and assessment of therapy and severity of pathological progression in multiple system atrophy. Because 2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy] benzoxazole could stain alpha-synuclein-containing glial cytoplasmic inclusions in post-mortem brains, we compared the carbon-11-labelled 2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy] benzoxazole positron emission tomography findings of eight multiple system atrophy cases to those of age-matched normal controls. The positron emission tomography data demonstrated high distribution volumes in the subcortical white matter (uncorrected P benzoxazole positron emission tomography is a promising surrogate marker for monitoring intracellular alpha-synuclein deposition in living brains.

  16. Alpha-synuclein promotes clathrin-mediated endocytosis of NMDA receptors in dopaminergic cells

    Institute of Scientific and Technical Information of China (English)

    Shun Yu; Furong Cheng; Xin Li; Yaohua Li; Tao Wang; Guangwei Liu; Andrius Baskys

    2012-01-01

    Loss of dopaminergic i a compensatory increase in nput to the striatum associated with Parkinson' s disease brings about glutamate release onto the dopaminergic cell bodies in the substantia nigra pars compacta (SNpc)[1] Glutamate over-activation of NMDA receptors on these cells can cause excitotoxicity and contribute to their further loss. NMDA receptor-mediated neuronal death is reduced by group I mGluR-mediated up-regulation of endocytosis protein RAB5B[2.3] Among proteins shown to interact with RAB5 proteins is a-synuclein

  17. Dose-dependent striatal changes in dopaminergic terminals and alpha-synuclein reactivity in a porcine model of progressive Parkinson’s disease

    DEFF Research Database (Denmark)

    Nielsen, Mette Slot; Glud, Andreas Nørgaard; Møller, Arne

    2011-01-01

    Parkinson disease (PD) is a common neurodegenerative disorder, resulting from a progressive dopaminergic neuron loss in the substantia nigra (SN). Alpha-synuclein positive neuronal inclusion bodies and progressive loss of dopaminergic striatal terminals is also well described in PD. Attempts......) or acute MPTP intoxication for 11 days (24 mg MPTP/day, n=2) and 9 weeks of recovery. Four pigs served as normal controls. Animals were euthanized with intracardial pentobarbital injections, transcardially perfused with 5 L 4% paraformaldehyde and the brains removed. The striatae and brain stems including...... the SN were paraffin embedded and immunohistochemically stained for tyrosine hydroxylase (TH) and alpha-synuclein. Stereological examination of the SN showed progressive nigral neuron loss with increased MPTP dosages. Occasional neuronal staining confined to the cytoplasm and cell membrane was observed...

  18. Neuroinflammation in Multiple System Atrophy: Response to and Cause of alpha-Synuclein Aggregation

    Directory of Open Access Journals (Sweden)

    Bruno eDi Marco Vieira

    2015-11-01

    Full Text Available Multiple system atrophy (MSA is a progressive neurodegenerative disease presenting with combinations of autonomic dysfunction, parkinsonism, cerebellar ataxia and/or pyramidal signs. Oligodendroglial cytoplasmic inclusions rich in α-synuclein (α-syn constitute the disease hallmark, accompanied by neuronal loss and activation of glial cells which indicate neuroinflammation. Recent studies demonstrate that α-syn may be released from degenerating neurons to mediate formation of abnormal inclusion bodies and to induce neuroinflammation which, interestingly, might also favour the formation of intracellular α-syn aggregates as a consequence of cytokine release and the shift to a pro-inflammatory environment. Here we critically review the relationships between α-syn and astrocytic and microglial activation in MSA to explore the potential of therapeutics which target neuroinflammation.

  19. Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    Directory of Open Access Journals (Sweden)

    Adrian Flierl

    Full Text Available Parkinson disease (PD is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs from a patient with Parkinson's disease carrying a genomic triplication of the SNCA gene (SNCA-Tri. Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this "stem cell pathology" could have a great impact on both quality and quantity of neuronal networks and could provide a

  20. The ER retention protein RER1 promotes alpha-synuclein degradation via the proteasome.

    Science.gov (United States)

    Park, Hyo-Jin; Ryu, Daniel; Parmar, Mayur; Giasson, Benoit I; McFarland, Nikolaus R

    2017-01-01

    Abnormal accumulation of α-synuclein (αSyn) has been linked to endoplasmic-reticulum (ER) stress, defective intracellular protein/vesicle trafficking, and cytotoxicity. Targeting factors involved in ER-related protein processing and trafficking may, therefore, be a key to modulating αSyn levels and associated toxicity. Recently retention in endoplasmic reticulum 1 (RER1) has been identified as an important ER retrieval/retention factor for Alzheimer's disease proteins and negatively regulates amyloid-β peptide levels. Here, we hypothesized that RER1 might also play an important role in retention/retrieval of αSyn and mediate levels. We expressed RER1 and a C-terminal mutant RER1Δ25, which lacks the ER retention/retrieval function, in HEK293 and H4 neuroglioma cells. RER1 overexpression significantly decreased levels of both wild type and A30P, A53T, and E46K disease causal mutants of αSyn, whereas the RER1Δ25 mutant had a significantly attenuated effect on αSyn. RER1 effects were specific to αSyn and had little to no effect on either βSyn or the Δ71-82 αSyn mutant, which both lack the NAC domain sequence critical for synuclein fibrillization. Tests with proteasomal and macroautophagy inhibitors further demonstrate that RER1 effects on αSyn are primarily mediated through the ubiquitin-proteasome system. RER1 also appears to interact with the ubiquitin ligase NEDD4. RER1 in human diseased brain tissues co-localizes with αSyn-positive Lewy bodies. Together, these findings provide evidence that RER1 is a novel and potential important mediator of elevated αSyn levels. Further investigation of the mechanism of RER1 and downstream effectors on αSyn may yield novel therapeutic targets for modulation in Parkinson disease and related synucleinopathies.

  1. Blood Plasma of Patients with Parkinson's Disease Increases Alpha-Synuclein Aggregation and Neurotoxicity

    Science.gov (United States)

    Wang, Peng; Li, Xin; Li, Xuran; Yang, Weiwei

    2016-01-01

    A pathological hallmark of Parkinson's disease (PD) is formation of Lewy bodies in neurons of the brain. This has been attributed to the spread of α-synuclein (α-syn) aggregates, which involves release of α-syn from a neuron and its reuptake by a neighboring neuron. We found that treatment with plasma from PD patients induced more α-syn phosphorylation and oligomerization than plasma from normal subjects (NS). Compared with NS plasma, PD plasma added to primary neuron cultures caused more cell death in the presence of extracellular α-syn. This was supported by the observations that phosphorylated α-syn oligomers entered neurons, rapidly increased accumulated thioflavin S-positive inclusions, and induced a series of metabolic changes that included activation of polo-like kinase 2, inhibition of glucocerebrosidase and protein phosphatase 2A, and reduction of ceramide levels, all of which have been shown to promote α-syn phosphorylation and aggregation. We also analyzed neurotoxicity of α-syn oligomers relative to plasma from different patients. Neurotoxicity was not related to age or gender of the patients. However, neurotoxicity was positively correlated with H&Y staging score. The modification in the plasma may promote spreading of α-syn aggregates via an alternative pathway and accelerate progression of PD. PMID:27965913

  2. Blood Plasma of Patients with Parkinson’s Disease Increases Alpha-Synuclein Aggregation and Neurotoxicity

    Directory of Open Access Journals (Sweden)

    Peng Wang

    2016-01-01

    Full Text Available A pathological hallmark of Parkinson’s disease (PD is formation of Lewy bodies in neurons of the brain. This has been attributed to the spread of α-synuclein (α-syn aggregates, which involves release of α-syn from a neuron and its reuptake by a neighboring neuron. We found that treatment with plasma from PD patients induced more α-syn phosphorylation and oligomerization than plasma from normal subjects (NS. Compared with NS plasma, PD plasma added to primary neuron cultures caused more cell death in the presence of extracellular α-syn. This was supported by the observations that phosphorylated α-syn oligomers entered neurons, rapidly increased accumulated thioflavin S-positive inclusions, and induced a series of metabolic changes that included activation of polo-like kinase 2, inhibition of glucocerebrosidase and protein phosphatase 2A, and reduction of ceramide levels, all of which have been shown to promote α-syn phosphorylation and aggregation. We also analyzed neurotoxicity of α-syn oligomers relative to plasma from different patients. Neurotoxicity was not related to age or gender of the patients. However, neurotoxicity was positively correlated with H&Y staging score. The modification in the plasma may promote spreading of α-syn aggregates via an alternative pathway and accelerate progression of PD.

  3. Nortriptyline inhibits aggregation and neurotoxicity of alpha-synuclein by enhancing reconfiguration of the monomeric form.

    Science.gov (United States)

    Collier, Timothy J; Srivastava, Kinshuk R; Justman, Craig; Grammatopoulous, Tom; Hutter-Paier, Birgit; Prokesch, Manuela; Havas, Daniel; Rochet, Jean-Christophe; Liu, Fang; Jock, Kevin; de Oliveira, Patrícia; Stirtz, Georgia L; Dettmer, Ulf; Sortwell, Caryl E; Feany, Mel B; Lansbury, Peter; Lapidus, Lisa; Paumier, Katrina L

    2017-10-01

    The pathology of Parkinson's disease and other synucleinopathies is characterized by the formation of intracellular inclusions comprised primarily of misfolded, fibrillar α-synuclein (α-syn). One strategy to slow disease progression is to prevent the misfolding and aggregation of its native monomeric form. Here we present findings that support the contention that the tricyclic antidepressant compound nortriptyline (NOR) has disease-modifying potential for synucleinopathies. Findings from in vitro aggregation and kinetics assays support the view that NOR inhibits aggregation of α-syn by directly binding to the soluble, monomeric form, and by enhancing reconfiguration of the monomer, inhibits formation of toxic conformations of the protein. We go on to demonstrate that NOR inhibits the accumulation, aggregation and neurotoxicity of α-syn in multiple cell and animal models. These findings suggest that NOR, a compound with established safety and efficacy for treatment of depression, may slow progression of α-syn pathology by directly binding to soluble, native, α-syn, thereby inhibiting pathological aggregation and preserving its normal functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Interplay between desolvation and secondary structure in mediating cosolvent and temperature induced alpha-synuclein aggregation

    Science.gov (United States)

    Anderson, V. L.; Webb, W. W.; Eliezer, D.

    2012-10-01

    Both increased temperature and moderate concentrations of fluorinated alcohols enhance aggregation of the Parkinson's disease-associated protein α-synuclein (αS). Here, we investigate the secondary structural rearrangements induced by heating and trifluoroethanol [TFE]. At low TFE concentrations, CD spectra feature a negative peak characteristic of disordered polypeptides near 200 nm and a slight shoulder around 220 nm suggesting some polyproline-II content. Upon heating, these peaks weaken, while a weak negative signal develops at 222 nm. At high TFE concentrations, the spectra show distinct minima at 208 and 222 nm, indicative of considerable α-helical structure, which diminish upon heating. We observe a crossover between the low-TFE and high-TFE behavior near 15% TFE, where we previously showed that a partially helical intermediate is populated. We postulate that the protein is well solvated by water at low TFE concentrations and by TFE at high TFE concentrations, but may become desolvated at the crossover point. We discuss the potential roles and interplay of desolvation and helical secondary structure in driving αS aggregation.

  5. An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Li [Department of Pathology, University of Washington, Seattle WA USA; Stewart, Tessandra [Department of Pathology, University of Washington, Seattle WA USA; Shi, Min [Department of Pathology, University of Washington, Seattle WA USA; Pottiez, Gwenael [Department of Pathology, University of Washington, Seattle WA USA; Dator, Romel [Department of Pathology, University of Washington, Seattle WA USA; Wu, Rui [Department of Pathology, University of Washington, Seattle WA USA; Department of Pathology, No. 3 Hospital of Beijing University, Beijing China; Aro, Patrick [Department of Pathology, University of Washington, Seattle WA USA; Schuster, Robert J. [Department of Pathology, University of Washington, Seattle WA USA; Ginghina, Carmen [Department of Pathology, University of Washington, Seattle WA USA; Pan, Catherine [Department of Pathology, University of Washington, Seattle WA USA; Gao, Yuqian [Pacific Northwest National Laboratory, Richland WA USA; Qian, Weijun [Pacific Northwest National Laboratory, Richland WA USA; Zabetian, Cyrus P. [Parkinson' s Disease Research and Geriatric Research, Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle WA USA; Department of Neurology, University of Washington School of Medicine, Seattle WA USA; Hu, Shu-Ching [Department of Neurology, University of Washington School of Medicine, Seattle WA USA; Quinn, Joseph F. [Department of Neurology, Oregon Health and Science University, Portland OR USA; Zhang, Jing [Department of Pathology, University of Washington, Seattle WA USA; Department of Pathology, Peking University Health Science Centre and Third Hospital, Beijing 100083 China

    2017-04-19

    Aim: The alpha-synuclein (α-syn) level in human cerebrospinal fluid (CSF), as measured by immunoassays, is promising as a Parkinson’s disease (PD) biomarker. However, the levels of total α-syn are inconsistent among studies with large cohorts and different measurement platforms. Total α-syn level also does not correlate with disease severity or progression. Here, we developed a highly sensitive Multiple Reaction Monitoring (MRM) method to measure absolute CSF α-syn peptide concentrations without prior enrichment or fractionation, aiming to discover new candidate biomarkers. Results: Six peptides covering 73% of protein sequence were reliably identified, and two were consistently quantified in cross-sectional and longitudinal cohorts. Absolute concentration of α-syn in human CSF was determined to be 2.1ng/mL. A unique α-syn peptide, TVEGAGSIAAATGFVK (81-96), displayed excellent correlation with previous immunoassay results in two independent PD cohorts (p < 0.001), correlated with disease severity, and its changes significantly tracked the disease progression longitudinally. Conclusions: An MRM assay to quantify human CSF α-syn was developed and optimized. Sixty clinical samples from cross-sectional and longitudinal PD cohorts were analyzed with this approach. Although further larger-scale validation is needed, the results suggest that α-syn peptide could serve as a promising biomarker in PD diagnosis and progression.

  6. Structured Regions of Alpha-synuclein Fibrils Include the Early Onset Parkinson's Disease Mutation Sites

    Energy Technology Data Exchange (ETDEWEB)

    Comellas Canal, Gemma; Lemkau, Luisel R.; Nieuwkoop, Andrew J.; Kloepper, Kathryn D.; Ladror, Daniel T.; Ebisu, Reika; Woods, Wendy S.; Lipton, Andrew S.; George, Julia M.; Rienstra, Chad M.

    2011-08-26

    Alpha-Synuclein (AS) fibrils constitute the major proteinaceous component of Lewy bodies (LBs), the pathological hallmark of Parkinson’s disease (PD) and other neurodegenerative diseases. Three single point mutations in the AS gene, as well as multiplication of the wild-type (WT) AS allele, have been previously identified in families with early-onset PD. Although AS fibrils have been the subject of intense study, critical details about their structure including the precise location of the B-strands and the extent of the core, the three-dimensional structure and the effects of the mutations—remain unknown. Here, we have used magic-angle spinning solid-state NMR spectroscopy to present a detailed characterization of the full-length WT AS fibrils. With improved sample preparations, isotopic labeling patterns and NMR experiments, we have confidently assigned more than 90% of the 13C and 15N backbone and sidechain chemical shifts of the detected residues from residue 39 to 97, and quantified the conformational dynamics throughout this region. Our results demonstrate that the core of AS fibrils extends with a repeated motif and that residues 30, 46 and 53-the early-onset PD mutant sites-are located in structured regions of AS fibrils.

  7. Comparison of structure and dynamics of micelle-bound human alpha-synuclein and Parkinson disease variants.

    Science.gov (United States)

    Ulmer, Tobias S; Bax, Ad

    2005-12-30

    Three point mutations (A30P, E46K, and A53T) as well as gene triplication genetically link the 140-residue protein alpha-synuclein (aS) to the development of Parkinson disease. Here, the structure and dynamics of micelle-bound aS(A30P) and aS(A53T) are described and compared with wild-type aS, in addition to describing the aS-micelle interaction. A53T is sensed only by directly adjacent residues and leaves the backbone structure and dynamics indistinguishable from the wild type. A30P interrupts one helix turn (Val26-Ala29) and destabilizes the preceding one. A shift in helix register following A30P disturbs the canonical succession of polar and hydrophobic residues for at least two turns. The shortened helix-N adopts a slightly higher helical content and is less bent, indicating that strain was present in the micelle-bound helix. In the vicinity of the A30P-induced perturbations, the underlying micelle environment has rearranged, but nevertheless all aS variants maintain similar interrelationships with the micelle. Moreover, aS-micelle immersion correlates well with fast and slow aS backbone dynamics, allowing a rare insight into protein-micelle interplay.

  8. Loss of β-glucocerebrosidase activity does not affect alpha-synuclein levels or lysosomal function in neuronal cells.

    Science.gov (United States)

    Dermentzaki, Georgia; Dimitriou, Evangelia; Xilouri, Maria; Michelakakis, Helen; Stefanis, Leonidas

    2013-01-01

    To date, a plethora of studies have provided evidence favoring an association between Gaucher disease (GD) and Parkinson's disease (PD). GD, the most common lysosomal storage disorder, results from the diminished activity of the lysosomal enzyme β-glucocerebrosidase (GCase), caused by mutations in the β-glucocerebrosidase gene (GBA). Alpha-synuclein (ASYN), a presynaptic protein, has been strongly implicated in PD pathogenesis. ASYN may in part be degraded by the lysosomes and may itself aberrantly impact lysosomal function. Therefore, a putative link between deficient GCase and ASYN, involving lysosomal dysfunction, has been proposed to be responsible for the risk for PD conferred by GBA mutations. In this current work, we aimed to investigate the effects of pharmacological inhibition of GCase on ASYN accumulation/aggregation, as well as on lysosomal function, in differentiated SH-SY5Y cells and in primary neuronal cultures. Following profound inhibition of the enzyme activity, we did not find significant alterations in ASYN levels, or any changes in the clearance or formation of its oligomeric species. We further observed no significant impairment of the lysosomal degradation machinery. These findings suggest that additional interaction pathways together with aberrant GCase and ASYN must govern this complex relation between GD and PD.

  9. Alpha-synuclein A53T mutation is not frequent on a sample of Brazilian Parkinson’s disease patients

    Directory of Open Access Journals (Sweden)

    Gabriela S. Longo

    2015-06-01

    Full Text Available Introduction The pathogenesis of Parkinson’s disease (PD involves both genetic susceptibility and environmental factors, with focus on the mutation in the alpha-synuclein gene (SNCA.Objective To analyse the polymorphism SNCA-A53T in patients with familial PD (FPD and sporadic PD (SPD.Method A total of 294 individuals were studied, regardless of sex and with mixed ethnicity. The study group with 154 patients with PD, and the control group included 140 individuals without PD. The genotyping of SNCA-A53T was performed by PCR/RFLP. Significance level was p < 0.05.Results Among all patients, 37 (24% had FPD and 117 (75.9% had SPD. The absence of SNCA-A53T mutation was observed in all individuals.Conclusion SPD is notably observed in patients. However, the SNCA-A53T mutation was absent in all individuals, which does not differ controls from patients. This fact should be confirmed in a Brazilian study case with a more numerous and older population.

  10. Structural characterization of copper(II) binding to alpha-synuclein: Insights into the bioinorganic chemistry of Parkinson's disease.

    Science.gov (United States)

    Rasia, Rodolfo M; Bertoncini, Carlos W; Marsh, Derek; Hoyer, Wolfgang; Cherny, Dmitry; Zweckstetter, Markus; Griesinger, Christian; Jovin, Thomas M; Fernández, Claudio O

    2005-03-22

    The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS-Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders.

  11. A rapid, semi-quantitative assay to screen for modulators of alpha-synuclein oligomerization ex vivo

    Directory of Open Access Journals (Sweden)

    Marion eDelenclos

    2016-01-01

    Full Text Available Alpha synuclein (αsyn aggregates are associated with the pathogenesis of Parkinson’s disease and others related disorders. Although modulation of αsyn aggregation is an attractive therapeutic target, new powerful methodologies are desperately needed to facilitate in vivo screening of novel therapeutics. Here we describe an in vivo rodent model with the unique ability to rapidly track αsyn-αsyn interactions and thus oligomerization using a bioluminescent protein complementation strategy that monitors spatial and temporal αsyn oligomerization ex vivo. We find that αsyn forms oligomers in vivo as early as 1 week after stereotactic AAV injection into rat substantia nigra. Strikingly, although abundant αsyn expression is also detected in striatum at one week, no αsyn oligomers are detected at this time point. By 4 weeks, oligomerization of αsyn is detected in both striatum and substantia nigra homogenates. Moreover, in a proof-of-principle experiment, the effect of a previously described Hsp90 inhibitor known to prevent αsyn oligomer formation, demonstrates the utility of this rapid and sensitive animal model to monitor αsyn oligomerization status in the rat brain.

  12. Leucine-rich repeat kinase 2 and alpha-synuclein: intersecting pathways in the pathogenesis of Parkinson's disease?

    Directory of Open Access Journals (Sweden)

    Civiero Laura

    2011-01-01

    Full Text Available Abstract Although Parkinson's disease (PD is generally a sporadic neurological disorder, the discovery of monogenic, hereditable forms of the disease has been crucial in delineating the molecular pathways that lead to this pathology. Genes responsible for familial PD can be ascribed to two categories based both on their mode of inheritance and their suggested biological function. Mutations in parkin, PINK1 and DJ-1 cause of recessive Parkinsonism, with a variable pathology often lacking the characteristic Lewy bodies (LBs in the surviving neurons. Intriguingly, recent findings highlight a converging role of all these genes in mitochondria function, suggesting a common molecular pathway for recessive Parkinsonism. Mutations in a second group of genes, encoding alpha-synuclein (α-syn and LRRK2, are transmitted in a dominant fashion and generally lead to LB pathology, with α-syn being the major component of these proteinaceous aggregates. In experimental systems, overexpression of mutant proteins is toxic, as predicted for dominant mutations, but the normal function of both proteins is still elusive. The fact that α-syn is heavily phosphorylated in LBs and that LRRK2 is a protein kinase, suggests that a link, not necessarily direct, exists between the two. What are the experimental data supporting a common molecular pathway for dominant PD genes? Do α-syn and LRRK2 target common molecules? Does LRRK2 act upstream of α-syn? In this review we will try to address these of questions based on the recent findings available in the literature.

  13. SMG1 identified as a regulator of Parkinson's disease-associated alpha-synuclein through siRNA screening.

    Directory of Open Access Journals (Sweden)

    Adrienne Henderson-Smith

    Full Text Available Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129 compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous in vitro and in vivo studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson's disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn. Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB and Parkinson's disease with dementia (PDD. These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.

  14. Cathepsin D expression level affects alpha-synuclein processing, aggregation, and toxicity in vivo

    Directory of Open Access Journals (Sweden)

    Cullen Valerie

    2009-02-01

    Full Text Available Abstract Background Elevated SNCA gene expression and intracellular accumulation of the encoded α-synuclein (aSyn protein are associated with the development of Parkinson disease (PD. To date, few enzymes have been examined for their ability to degrade aSyn. Here, we explore the effects of CTSD gene expression, which encodes the lysosomal protease cathepsin D (CathD, on aSyn processing. Results Over-expression of human CTSD cDNA in dopaminergic MES23.5 cell cultures induced the marked proteolysis of exogenously expressed aSyn proteins in a dose-dependent manner. Unexpectedly, brain extractions, Western blotting and ELISA quantification revealed evidence for reduced levels of soluble endogenous aSyn in ctsd knock-out mice. However, these CathD-deficient mice also contained elevated levels of insoluble, oligomeric aSyn species, as detected by formic acid extraction. In accordance, immunohistochemical studies of ctsd-mutant brain from mice, sheep and humans revealed selective synucleinopathy-like changes that varied slightly among the three species. These changes included intracellular aSyn accumulation and formation of ubiquitin-positive inclusions. Furthermore, using an established Drosophila model of human synucleinopathy, we observed markedly enhanced retinal toxicity in ctsd-null flies. Conclusion We conclude from these complementary investigations that: one, CathD can effectively degrade excess aSyn in dopaminergic cells; two, ctsd gene mutations result in a lysosomal storage disorder that includes microscopic and biochemical evidence of aSyn misprocessing; and three, CathD deficiency facilitates aSyn toxicity. We therefore postulate that CathD promotes 'synucleinase' activity, and that enhancing its function may lower aSyn concentrations in vivo.

  15. Wildtype and A30P mutant alpha-synuclein form different fibril structures.

    Directory of Open Access Journals (Sweden)

    Søren Bang Nielsen

    Full Text Available Parkinson's Disease (PD is a neurodegenerative movement disorder affecting millions of people worldwide. One of the key players in the development of the disease is the protein α-synuclein (aSN, which aggregates in the brain of PD patients. The aSN mutant A30P has been reported to cause early-onset familial PD and shows different aggregation behavior compared to wt aSN. Here we use a multidisciplinary approach to compare the aggregation process of wt and A30P aSN. In agreement with previous studies, we observe an initial lag phase followed by a continuous structural development of fibrils until reaching an apparent monomer-aggregate equilibrium state and a plateau in Thioflavin T (ThT fluorescence intensity. However, at later timepoints A30P shows greater propensity than αSN wt to form dense bundled fibril networks. Combining small angle x-ray scattering, x-ray fibre diffraction and linear dichroism, we demonstrate that while the microscopic structure of the individual fibril essentially remains constant throughout the experiment, the formation of dense A30P fibril networks occur through a continuous assembly pathway while the formation of less dense wt fibril networks with fewer contact points follows a continuous path during the elongation phase and a second rearrangement phase after reaching the ThT fluorescence plateau. Our work thus highlights that structural rearrangements proceed beyond the plateau in ThT-based monitoring of the fibrillation process, and the density and morphology of the resulting fibril networks is highly dependent on the aSN form studied.

  16. Alpha-synuclein mRNA expression in oligodendrocytes in MSA.

    Science.gov (United States)

    Asi, Yasmine T; Simpson, Julie E; Heath, Paul R; Wharton, Stephen B; Lees, Andrew J; Revesz, Tamas; Houlden, Henry; Holton, Janice L

    2014-06-01

    Multiple system atrophy (MSA) is a progressive neurodegenerative disease presenting clinically with parkinsonian, cerebellar, and autonomic features. α-Synuclein (αsyn), encoded by the gene SNCA, is the main constituent of glial cytoplasmic inclusion (GCI) found in oligodendrocytes in MSA, but the methods of its accumulation have not been established. The aim of this study is to investigate alterations in regional and cellular SNCA mRNA expression in MSA as a possible substrate for GCI formation. Quantitative reverse transcription polymerase chain reaction (qPCR) was performed on postmortem brain samples from 15 MSA, 5 IPD, and 5 control cases to investigate regional expression in the frontal and occipital regions, dorsal putamen, pontine base, and cerebellum. For cellular expression analysis, neurons and oligodendrocytes were isolated by laser-capture microdissection from five MSA and five control cases. SNCA mRNA expression was not significantly different between the MSA, IPD and control cases in all regions (multilevel model, P = 0.14). After adjusting for group effect, the highest expression was found in the occipital cortex while the lowest was in the putamen (multilevel model, P MSA oligodendrocytes expressed more SNCA than control oligodendrocytes and expression in MSA neurons was slightly lower than that in controls, however, these results did not reach statistical significance. We have demonstrated regional variations in SNCA expression, which is higher in cortical than subcortical regions. This study is the first to demonstrate SNCA mRNA expression by oligodendrocytes in human postmortem tissue using qPCR and, although not statistically significant, could suggest that this may be increased in MSA compared to controls.

  17. Molecular cloning, characterization and developmental expression of porcine β-synuclein

    DEFF Research Database (Denmark)

    Larsen, Knud; Frandsen, Pernille Munk; Madsen, Lone Bruhn

    2010-01-01

    The synuclein family includes three known proteins: alpha-synuclein, beta-synuclein and gamma-synuclein. beta-Synuclein inhibits the aggregation of alpha-synuclein, a protein involved in Parkinson's disease. We have cloned and characterized the cDNA sequence for porcine beta-synuclein (SNCB) from...... development. Radiation hybrid mapping data indicate that the porcine SNCB maps to the q arm of chromosome 2 (2q21-22). The subcellular localization of recombinant porcine beta-synuclein was determined in three different cell types and shown to be cytoplasmic. Udgivelsesdato: March...

  18. Alpha-synuclein ferrireductase activity is detectible in vivo, is altered in Parkinson's disease and increases the neurotoxicity of DOPAL.

    Science.gov (United States)

    McDowall, Jennifer S; Ntai, Ioanna; Honeychurch, Kevin C; Hart, John P; Colin, Philippe; Schneider, Bernard L; Brown, David R

    2017-08-12

    The normal cellular role of α-synuclein is of potential importance in understanding diseases in which an aggregated form of the protein has been implicated. A potential loss or change in the normal function of α-synuclein could play a role in the aetiology of diseases such as Parkinson's disease. Recently, it has been suggested that α-synuclein could cause the enzymatic reduction of iron and a cellular increase in Fe(II) levels. Experiments were carried out to determine if such activity could be measured in vivo. Experiments with rats overexpressing human α-synuclein in nigral dopaminergic neurons demonstrated a correlation between α-synuclein expression and ferrireductase activity. Furthermore, studies on tissue from Parkinson's disease patient brains showed a significant decrease in ferrireductase activity, possibly due to deposition of large amounts of inactive protein. Cellular studies suggest that increase ferrireductase activity results in increased levels of dopamine metabolites and increased sensitivity to the toxicity of DOPAL. These findings demonstrate that α-synuclein ferrireductase activity is present in vivo and its alteration may play a role in neuron loss in disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The mechanism of sirtuin 2–mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease

    Science.gov (United States)

    Francelle, Laetitia; Pinho, Raquel; Szegö, Éva M.; Martinho, Renato; Munari, Francesca; Lázaro, Diana F.; Moniot, Sébastien; Guerreiro, Patrícia; Fonseca, Luis; Marijanovic, Zrinka; Antas, Pedro; Gerhardt, Ellen; Enguita, Francisco Javier; Fauvet, Bruno; Penque, Deborah; Pais, Teresa Faria; Tong, Qiang; Becker, Stefan; Kügler, Sebastian; Lashuel, Hilal Ahmed; Steegborn, Clemens; Zweckstetter, Markus; Outeiro, Tiago Fleming

    2017-01-01

    Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies. PMID:28257421

  20. Bond scission cross sections for alpha-particles in cellulose nitrate (LR115)

    CERN Document Server

    Barillon, R; Chambaudet, A; Katz, R; Stoquert, J P; Pape, A

    1999-01-01

    Chemical damage created by alpha-particles in cellulose nitrate (LR115) have been studied by infrared spectroscopy. This technique enables identifying the sensitive bonds and giving an order of magnitude of their scission cross sections for given alpha-particle energies. The high cross sections observed suggest a new description of the track etch velocity in this material.

  1. Charge state dependent fragmentation of gaseous [alpha]-synuclein cations via ion trap and beam-type collisional activation

    Science.gov (United States)

    Chanthamontri, Chamnongsak; Liu, Jian; McLuckey, Scott A.

    2009-06-01

    Ions derived from nano-electrospray ionization (nano-ESI) of [alpha]-synuclein, a 14.5 kDa, 140 amino acid residue protein that is a major component of the Lewy bodies associated with Parkinson's disease, have been subjected to ion trap and beam-type collisional activation. The former samples products from fragmentation at rates generally lower than 100 s-1 whereas the latter samples products from fragmentation at rates generally greater than 103 s-1. A wide range of protein charge states spanning from as high as [M+17H]17+ to as low as [M+4H]4+ have been formed either directly from nano-ESI or via ion/ion proton transfer reactions involving the initially formed protein cations and have been subjected to both forms of collision-induced dissociation (CID). The extent of sequence information (i.e., number of distinct amide bond cleavages) available from either CID method was found to be highly sensitive to protein precursor ion charge state. Furthermore, the relative contributions of the various competing dissociation channels were also dependent upon precursor ion charge state. The qualitative trends in the changes in extent of amide bond cleavages and identities of bonds cleaved with precursor ion charge state were similar for two forms of CID. However, for every charge state examined, roughly twice the primary sequence information resulted from beam-type CID relative to ion trap CID. For example, evidence for cleavage of 86% of the protein amide bonds was observed for the [M+9H]9+ precursor ion using beam-type CID whereas 41% of the bonds were cleaved for the same precursor ion using ion trap CID. The higher energies required to drive fragmentation reactions at rates necessary to observe products in the beam experiment access more of the structurally informative fragmentation channels, which has important implications for whole protein tandem mass spectrometry.

  2. Novel oligodendroglial alpha synuclein viral vector models of multiple system atrophy: studies in rodents and nonhuman primates.

    Science.gov (United States)

    Mandel, Ronald J; Marmion, David J; Kirik, Deniz; Chu, Yaping; Heindel, Clifford; McCown, Thomas; Gray, Steven J; Kordower, Jeffrey H

    2017-06-16

    Multiple system atrophy (MSA) is a horrible and unrelenting neurodegenerative disorder with an uncertain etiology and pathophysiology. MSA is a unique proteinopathy in which alpha-synuclein (α-syn) accumulates preferentially in oligodendroglia rather than neurons. Glial cytoplasmic inclusions (GCIs) of α-syn are thought to elicit changes in oligodendrocyte function, such as reduced neurotrophic support and demyelination, leading to neurodegeneration. To date, only a murine model using one of three promoters exist to study this disease. We sought to develop novel rat and nonhuman primate (NHP) models of MSA by overexpressing α-syn in oligodendroglia using a novel oligotrophic adeno-associated virus (AAV) vector, Olig001. To establish tropism, rats received intrastriatal injections of Olig001 expressing GFP. Histological analysis showed widespread expression of GFP throughout the striatum and corpus callosum with >95% of GFP+ cells co-localizing with oligodendroglia and little to no expression in neurons or astrocytes. We next tested the efficacy of this vector in rhesus macaques with intrastriatal injections of Olig001 expressing GFP. As in rats, we observed a large number of GFP+ cells in gray matter and white matter tracts of the striatum and the corpus callosum, with 90-94% of GFP+ cells co-localizing with an oligodendroglial marker. To evaluate the potential of our vector to elicit MSA-like pathology in NHPs, we injected rhesus macaques intrastriatally with Olig001 expressing the α-syn transgene. Histological analysis 3-months after injection demonstrated widespread α-syn expression throughout the striatum as determined by LB509 and phosphorylated serine-129 α-syn immunoreactivity, all of which displayed as tropism similar to that seen with GFP. As in MSA, Olig001-α-syn GCIs in our model were resistant to proteinase K digestion and caused microglial activation. Critically, demyelination was observed in the white matter tracts of the corpus callosum and

  3. Propagation of alpha-synuclein pathology: hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies.

    Science.gov (United States)

    Uchihara, Toshiki; Giasson, Benoit I

    2016-01-01

    Progressive aggregation of alpha-synuclein (αS) through formation of amorphous pale bodies to mature Lewy bodies or in neuronal processes as Lewy neurites may be the consequence of conformational protein changes and accumulations, which structurally represents "molecular template". Focal initiation and subsequent spread along anatomically connected structures embody "structural template". To investigate the hypothesis that both processes might be closely associated and involved in the progression of αS pathology, which can be observed in human brains, αS amyloidogenic precursors termed "seeds" were experimentally injected into the brain or peripheral nervous system of animals. Although these studies showed that αS amyloidogenic seeds can induce αS pathology, which can spread in the nervous system, the findings are still not unequivocal in demonstrating predominant transsynaptic or intraneuronal spreads either in anterograde or retrograde directions. Interpretation of some of these studies is further complicated by other concurrent aberrant processes including neuroimmune activation, injury responses and/or general perturbation of proteostasis. In human brain, αS deposition and neuronal degeneration are accentuated in distal axon/synapse. Hyperbranching of axons is an anatomical commonality of Lewy-prone systems, providing a structural basis for abundance in distal axons and synaptic terminals. This neuroanatomical feature also can contribute to such distal accentuation of vulnerability in neuronal demise and the formation of αS inclusion pathology. Although retrograde progression of αS aggregation in hyperbranching axons may be a consistent feature of Lewy pathology, the regional distribution and gradient of Lewy pathology are not necessarily compatible with a predictable pattern such as upward progression from lower brainstem to cerebral cortex. Furthermore, "focal Lewy body disease" with the specific isolated involvement of autonomic, olfactory or cardiac

  4. 表达alpha-synuclein A53T神经干细胞系的建立%Establishment of murine neural stem cells with expression of alpha-synuclein A53T

    Institute of Scientific and Technical Information of China (English)

    沈雁飞; 柳明杰

    2013-01-01

    目的 建立表达alpha-synuleinA53T突变体的神经干细胞.方法 采用Xho Ⅰ/Hpa Ⅰ双酶切paSynA53T-DsRed,将含SynA53T-DsRed片段连接到pBudCE4.1-1X-aSynA53T-DsRed的Xho Ⅰ/Pme Ⅰ位点构建pBudCE4.1-2X-aSynA53T-DsRed质粒.利用Lipofectamine2000介导转染2μg的pBudCE4.1-2X-aSynA53T-DsRed到鼠源性神经干细胞(mNSCs)后24h和72 h观察转染结果.结果 成功构建重组质粒pBudCE4.1-2X-aSynA53T-DsRed,转染24h后其表达率达到(57±3.52)%,72 h的表达率达到了(27.14±3.21)%.结论 通过质粒重组及转染技术,成功获得了表达aSynA53T的神经干细胞,为揭示α-synA53T导致帕金森氏症的病理过程提供了平台.%Objective To produce murine neural stem cells (mNSCs)with expression of alpha-synuclein A53T.Methods The Xho Ⅰ/Hpa Ⅰ fragment of SynA53T-DsRed was released from paSynA53T-DsRed and cloned into Xho Ⅰ/Pme Ⅰ sites of pBudCE4.1-1X-aSynA53T-DsRed plasmid.Then 2 μg of recombinant plasmid was transfected into murine neural stem cells to establish the mNSCs with overexpression of aSynA53T.Results The pBudCE4.1-2X-aSynA53T-DsRed plasmid containing double aSynA53T expressing inserts downstream of CMV and EF-1αpromoters was successfully constructed.Expression levels of the recombinant plasmid reached to (57 ±3.52)% and (27.14 ±3.21)% after 24 h and 72 h transfection,respectively.Conclusions A recombinant plasmid pBudCF4.1-2X-aSynA53T-DsRed and mNSCs with expression of aSynA53T should be useful for the investigation of the pathogenesis of Parkinson's disease (PD) and development of drug for treatment of PD.

  5. At low concentrations, 3,4-dihydroxyphenylacetic acid (DOPAC) binds non-covalently to alpha-synuclein and prevents its fibrillation.

    Science.gov (United States)

    Zhou, Wenbo; Gallagher, Amy; Hong, Dong-Pyo; Long, Chunmei; Fink, Anthony L; Uversky, Vladimir N

    2009-05-01

    Several studies have shown that catecholamines can inhibit the fibrillation of alpha-synuclein (alpha-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of alpha-Syn, due to non-covalent binding of DOPAC to alpha-Syn monomer. Intriguingly, the presence of alpha-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of alpha-Syn, probably due to the H(2)O(2) production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the alpha-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the alpha-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of alpha-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify alpha-Syn covalently, probably due to the less steric hindrance in the Michael addition.

  6. Dopamine Cytotoxicity Involves Both Oxidative and Nonoxidative Pathways in SH-SY5Y Cells: Potential Role of Alpha-Synuclein Overexpression and Proteasomal Inhibition in the Etiopathogenesis of Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Kalpita Banerjee

    2014-01-01

    Full Text Available Background. The cytotoxic effects of dopamine (DA on several catecholaminergic cell lines involve DA oxidation products like reactive oxygen species (ROS and toxic quinones and have implications in the pathogenesis of sporadic Parkinson's disease (PD. However, many molecular details are yet to be elucidated, and the possible nonoxidative mechanism of dopamine cytotoxicity has not been studied in great detail. Results. Cultured SH-SY5Y cells treated with DA (up to 400 μM or lactacystin (5 μM or DA (400 μM plus N-acetylcysteine (NAC, 2.5 mM for 24 h are processed accordingly to observe the cell viability, mitochondrial dysfunctions, oxidative stress parameters, proteasomal activity, expression of alpha-synuclein gene, and intracellular accumulation of the protein. DA causes mitochondrial dysfunction and extensive loss of cell viability partially inhibited by NAC, potent inhibition of proteasomal activity marginally prevented by NAC, and overexpression with accumulation of intracellular alpha-synuclein partially preventable by NAC. Under similar conditions of incubation, NAC completely prevents enhanced production of ROS and increased formation of quinoprotein adducts in DA-treated SH-SY5Y cells. Separately, proteasomal inhibitor lactacystin causes accumulation of alpha-synuclein as well as mitochondrial dysfunction and cell death. Conclusions. DA cytotoxicity includes both oxidative and nonoxidative modes and may involve overexpression and accumulation of alpha-synuclein as well as proteasomal inhibition.

  7. Dose-dependent striatal changes in dopaminergic terminals and alpha-synuclein reactivity in a porcine model of progressive Parkinson’s disease

    DEFF Research Database (Denmark)

    Nielsen, Mette Slot; Glud, Andreas Nørgaard; Møller, Arne

    2011-01-01

    to discover effective compounds halting PD progression have so far failed in clinical trials, perhaps because current animal models do not imitate the neuropathological progression of PD well enough. We recently established a progressive large animal PD model in Göttingen minipigs based on chronic infusion...... and increased intensity in the neuronal staining. We conclude that chronic infusion of MPTP in this large animal PD model shows neuropathological changes resembling those seen in the clinical state of PD. Animals receiving 18 mg MPTP/day for 11 weeks thus showed a decrease in nigral TH-positive neuron number...... and striatal terminals, intense striatal alpha-synuclein staining and pathology in the locus coeruleus. Use of this model thus holds promise for future neuroprotective studies in PD....

  8. Microglia acquire distinct activation profiles depending on the degree of alpha-synuclein neuropathology in a rAAV based model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Vanesa Sanchez-Guajardo

    Full Text Available Post-mortem analysis of brains from Parkinson's disease (PD patients strongly supports microglia activation and adaptive immunity as factors contributing to disease progression. Such responses may be triggered by alpha-synuclein (alpha-syn, which is known to be the main constituent of the aggregated proteins found in Lewy bodies in the brains of PD patients. To investigate this we used a recombinant viral vector to express human alpha-syn in rat midbrain at levels that induced neuronal pathology either in the absence or the presence of dopaminergic cell death, thereby mimicking early or late stages of the disease. Microglia activation was assessed by stereological quantification of Mac1+ cells, as well as the expression patterns of CD68 and MCH II. In our study, when alpha-syn induced neuronal pathology but not cell death, a fast transient increase in microglia cell numbers resulted in the long-term induction of MHC II+ microglia, denoting antigen-presenting ability. On the other hand, when alpha-syn induced both neuronal pathology and cell death, there was a delayed increase in microglia cell numbers, which correlated with long-lasting CD68 expression and a morphology reminiscent of peripheral macrophages. In addition T-lymphocyte infiltration, as judged by the presence of CD4+ and CD8+ cells, showed distinct kinetics depending on the degree of neurodegeneration, and was significantly higher when cell death occurred. We have thus for the first time shown that the microglial response differs depending on whether alpha-syn expression results on cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, our data suggest that the microglial response is modulated by early events related to alpha-syn expression in substantia nigra and persists at the long term.

  9. A small-angle X-ray scattering study of alpha-synuclein from human red blood cells.

    Science.gov (United States)

    Araki, Katsuya; Yagi, Naoto; Nakatani, Rie; Sekiguchi, Hiroshi; So, Masatomo; Yagi, Hisashi; Ohta, Noboru; Nagai, Yoshitaka; Goto, Yuji; Mochizuki, Hideki

    2016-07-29

    α-synuclein (α-syn) is the main component of Lewy bodies, which are neuropathological hallmarks of patients with Parkinson's disease. As it has been controversial whether human α-syn from erythrocytes exists as a tetramer under physiological conditions, we tried solving this issue by the small-angle X-ray solution scattering method. Under two different conditions (high ionic strength with a Tris buffer and low ionic strength with an ammonium acetate buffer), no evidence was found for the presence of tetramer. When comparing erythrocyte and recombinant α-syn molecules, we found no significant difference of the molecular weight and the secondary structure although the buffer conditions strongly affect the radius of gyration of the protein. The results indicate that, even though a stable tetramer may not be formed, conformation of α-syn depends much on its environment, which may be the reason for its tendency to aggregate in cells.

  10. Structural variation of alpha-synuclein with temperature by a coarse-grained approach with knowledge-based interactions

    Directory of Open Access Journals (Sweden)

    Peter Mirau

    2015-09-01

    Full Text Available Despite enormous efforts, our understanding the structure and dynamics of α-synuclein (ASN, a disordered protein (that plays a key role in neurodegenerative disease is far from complete. In order to better understand sequence-structure-property relationships in α-SYNUCLEIN we have developed a coarse-grained model using knowledge-based residue-residue interactions and used it to study the structure of free ASN as a function of temperature (T with a large-scale Monte Carlo simulation. Snapshots of the simulation and contour contact maps show changes in structure formation due to self-assembly as a function of temperature. Variations in the residue mobility profiles reveal clear distinction among three segments along the protein sequence. The N-terminal (1-60 and C-terminal (96-140 regions contain the least mobile residues, which are separated by the higher mobility non-amyloid component (NAC (61-95. Our analysis of the intra-protein contact profile shows a higher frequency of residue aggregation (clumping in the N-terminal region relative to that in the C-terminal region, with little or no aggregation in the NAC region. The radius of gyration (Rg of ASN decays monotonically with decreasing the temperature, consistent with the finding of Allison et al. (JACS, 2009. Our analysis of the structure function provides an insight into the mass (N distribution of ASN, and the dimensionality (D of the structure as a function of temperature. We find that the globular structure with D ≈ 3 at low T, a random coil, D ≈ 2 at high T and in between (2 ≤ D ≤ 3 at the intermediate temperatures. The magnitudes of D are in agreement with experimental estimates (J. Biological Chem 2002.

  11. Structural variation of alpha-synuclein with temperature by a coarse-grained approach with knowledge-based interactions

    Science.gov (United States)

    Mirau, Peter; Farmer, B. L.; Pandey, R. B.

    2015-09-01

    Despite enormous efforts, our understanding the structure and dynamics of α-synuclein (ASN), a disordered protein (that plays a key role in neurodegenerative disease) is far from complete. In order to better understand sequence-structure-property relationships in α-SYNUCLEIN we have developed a coarse-grained model using knowledge-based residue-residue interactions and used it to study the structure of free ASN as a function of temperature (T) with a large-scale Monte Carlo simulation. Snapshots of the simulation and contour contact maps show changes in structure formation due to self-assembly as a function of temperature. Variations in the residue mobility profiles reveal clear distinction among three segments along the protein sequence. The N-terminal (1-60) and C-terminal (96-140) regions contain the least mobile residues, which are separated by the higher mobility non-amyloid component (NAC) (61-95). Our analysis of the intra-protein contact profile shows a higher frequency of residue aggregation (clumping) in the N-terminal region relative to that in the C-terminal region, with little or no aggregation in the NAC region. The radius of gyration (Rg) of ASN decays monotonically with decreasing the temperature, consistent with the finding of Allison et al. (JACS, 2009). Our analysis of the structure function provides an insight into the mass (N) distribution of ASN, and the dimensionality (D) of the structure as a function of temperature. We find that the globular structure with D ≈ 3 at low T, a random coil, D ≈ 2 at high T and in between (2 ≤ D ≤ 3) at the intermediate temperatures. The magnitudes of D are in agreement with experimental estimates (J. Biological Chem 2002).

  12. Nitrates

    Science.gov (United States)

    ... Blockers Angiotensin-Converting Enzyme (ACE) Inhibitors Antiarrhythmics Anticoagulants Antiplatelet Therapy Aspirin Beta-Blockers Blood Thinners Calcium Channel Blockers Digitalis Medicines Diuretics Inotropic Agents Statins, Cholesterol-Lowering Medicines Nitrates Disclaimer The information ...

  13. Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein

    Directory of Open Access Journals (Sweden)

    Severinovskaya O. V.

    2014-05-01

    Full Text Available Aim. To study the -synuclein (ASN aggregates of different structural origin, namely amyloid fibrils and spherical oligomers, in comparison with a native protein. Methods. MALDI TOF mass spectrometry and atomic for- ce microscopy (AFM. Results. The mass spectra of native and fibrillar ASN have similar character, i. e. they are characterized by the well pronounced peak of protein molecular ion, the low molecular weight associates, and rather low contain of fragmentation products. The spectrum of oligomeric aggregate is characterized by the high contain of fragmentation products, low intensity of protein molecular ion and the absence of peaks of associates. Conclusions. The difference between the spectra of fibrillar and oligomeric ASN could be explained, first, by the different content of the «residual» monomeric ASN and the protein degradation products in the studied samples, and, second, by the different structure-depended mechanisms of the protein degradation induced by the laser ionization. We suggested that the MALDI-TOF mass spectroscopy is a method useful for the investigation of ASN aggregation and characterization of its high order self-associates; besides, there is an interest in estimating the potency of the MALDI-TOF for the analysis of aggregation of various amyloidogenic proteins.

  14. Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in an AAV-Based Rat Genetic Model of Parkinson's Disease.

    Directory of Open Access Journals (Sweden)

    Abid Oueslati

    Full Text Available Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM, may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn in the substantia nigra of an AAV-based rat genetic model of Parkinson's disease (PD. In this model, daily exposure of both sides of the rat's head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies.

  15. Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in an AAV-Based Rat Genetic Model of Parkinson’s Disease

    Science.gov (United States)

    Oueslati, Abid; Lovisa, Blaise; Perrin, John; Wagnières, Georges; van den Bergh, Hubert; Tardy, Yanik; Lashuel, Hilal A.

    2015-01-01

    Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson’s disease (PD). In this model, daily exposure of both sides of the rat’s head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies. PMID:26484876

  16. Inhibition by Multifunctional Magnetic Nanoparticles Loaded with Alpha-Synuclein RNAi Plasmid in a Parkinson's Disease Model

    Science.gov (United States)

    Niu, Shuiqin; Zhang, Ling-Kun; Zhang, Li; Zhuang, Siyi; Zhan, Xiuyu; Chen, Wu-Ya; Du, Shiwei; Yin, Liang; You, Rong; Li, Chu-Hua; Guan, Yan-Qing

    2017-01-01

    Lewy bodies are considered as the main pathological characteristics of Parkinson's disease (PD). The major component of Lewy bodies is α-synuclein (α-syn). The use of gene therapy that targeting and effectively interfere with the expression of α-syn in neurons has received tremendous attention. In this study, we used magnetic Fe3O4 nanoparticles coated with oleic acid molecules as a nano-carrier. N-isopropylacrylamide derivative (NIPAm-AA) was photo-immobilized onto the oleic acid molecules, and shRNA (short hairpin RNA) was absorbed. The same method was used to absorb nerve growth factor (NGF) to NIPAm-AA to specifically promote neuronal uptake via NGF receptor-mediated endocytosis. Additionally, shRNA plasmid could be released into neurons because of the temperature and pH sensitivity of NIPAm-AA interference with α-syn synthesis. We investigated apoptosis in neurons with abrogated α-syn expression in vitro and in vivo. The results demonstrated that multifunctional superparamagnetic nanoparticles carrying shRNA for α-syn could provide effective repair in a PD model. PMID:28042339

  17. Cerebrospinal Fluid Amyloid β1-42, Tau, and Alpha-Synuclein Predict the Heterogeneous Progression of Cognitive Dysfunction in Parkinson's Disease.

    Science.gov (United States)

    Kang, Ju-Hee

    2016-05-01

    Parkinson's disease (PD) is a neurodegenerative disease with heterogeneous pathological and clinical features. Cognitive dysfunction, a frequent non-motor complication, is a risk factor for poor prognosis and shows inter-individual variation in its progression. Of the clinical studies performed to identify biomarkers of PD progression, the Parkinson's Progression Markers Initiative (PPMI) study is the largest study that enrolled drug-naïve and very early stage PD patients. The baseline characteristics of the PPMI cohort were recently published. The diagnostic utility of cerebrospinal fluid (CSF) biomarkers, including alpha-synuclein (α-syn), total tau, phosphorylated tau at Thr181, and amyloid β1-42, was not satisfactory. However, the baseline data on CSF biomarkers in the PPMI study suggested that the measurement of the CSF biomarkers enables the prediction of future cognitive decline in PD patients, which was consistent with previous studies. To prove the hypothesis that the interaction between Alzheimer's pathology and α-syn pathology is important to the progression of cognitive dysfunction in PD, longitudinal observational studies must be followed. In this review, the neuropathological nature of heterogeneous cognitive decline in PD is briefly discussed, followed by a summarized interpretation of baseline CSF biomarkers derived from the data in the PPMI study. The combination of clinical, biochemical, genetic and imaging biomarkers of PD constitutes a feasible strategy to predict the heterogeneous progression of PD.

  18. TRAF6 promotes atypical ubiquitination of mutant DJ-1 and alpha-synuclein and is localized to Lewy bodies in sporadic Parkinson's disease brains.

    Science.gov (United States)

    Zucchelli, Silvia; Codrich, Marta; Marcuzzi, Federica; Pinto, Milena; Vilotti, Sandra; Biagioli, Marta; Ferrer, Isidro; Gustincich, Stefano

    2010-10-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the Substantia Nigra and the formation of ubiquitin- and alpha-synuclein (aSYN)-positive cytoplasmic inclusions called Lewy bodies (LBs). Although most PD cases are sporadic, families with genetic mutations have been found. Mutations in PARK7/DJ-1 have been associated with autosomal recessive early-onset PD, while missense mutations or duplications of aSYN (PARK1, PARK4) have been linked to dominant forms of the disease. In this study, we identify the E3 ubiquitin ligase tumor necrosis factor-receptor associated factor 6 (TRAF6) as a common player in genetic and sporadic cases. TRAF6 binds misfolded mutant DJ-1 and aSYN. Both proteins are substrates of TRAF6 ligase activity in vivo. Interestingly, rather than conventional K63 assembly, TRAF6 promotes atypical ubiquitin linkage formation to both PD targets that share K6-, K27- and K29- mediated ubiquitination. Importantly, TRAF6 stimulates the accumulation of insoluble and polyubiquitinated mutant DJ-1 into cytoplasmic aggregates. In human post-mortem brains of PD patients, TRAF6 protein colocalizes with aSYN in LBs. These results reveal a novel role for TRAF6 and for atypical ubiquitination in PD pathogenesis.

  19. Similar patterns of mitochondrial vulnerability and rescue induced by genetic modification of alpha-synuclein, parkin, and DJ-1 in Caenorhabditis elegans.

    Science.gov (United States)

    Ved, Rina; Saha, Shamol; Westlund, Beth; Perier, Celine; Burnam, Lucinda; Sluder, Anne; Hoener, Marius; Rodrigues, Cecilia M P; Alfonso, Aixa; Steer, Clifford; Liu, Leo; Przedborski, Serge; Wolozin, Benjamin

    2005-12-30

    How genetic and environmental factors interact in Parkinson disease is poorly understood. We have now compared the patterns of vulnerability and rescue of Caenorhabditis elegans with genetic modifications of three different genetic factors implicated in Parkinson disease (PD). We observed that expressing alpha-synuclein, deleting parkin (K08E3.7), or knocking down DJ-1 (B0432.2) or parkin produces similar patterns of pharmacological vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than nontransgenic nematodes to mitochondrial complex I inhibitors, including rotenone, fenperoximate, pyridaben, or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity to paraquat, sodium azide, divalent metal ions (Fe(II) or Cu(II)), or etoposide compared with the nontransgenic nematodes. Each of the PD-related lines was also partially rescued by the antioxidant probucol, the mitochondrial complex II activator, D-beta-hydroxybutyrate, or the anti-apoptotic bile acid tauroursodeoxycholic acid. Complete protection in all lines was achieved by combining d-beta-hydroxybutyrate with tauroursodeoxycholic acid but not with probucol. These results show that diverse PD-related genetic modifications disrupt the mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD.

  20. Autophagy down regulates pro-inflammatory mediators in BV2 microglial cells and rescues both LPS and alpha-synuclein induced neuronal cell death

    Science.gov (United States)

    Bussi, Claudio; Ramos, Javier Maria Peralta; Arroyo, Daniela S.; Gaviglio, Emilia A.; Gallea, Jose Ignacio; Wang, Ji Ming; Celej, Maria Soledad; Iribarren, Pablo

    2017-01-01

    Autophagy is a fundamental cellular homeostatic mechanism, whereby cells autodigest parts of their cytoplasm for removal or turnover. Neurodegenerative disorders are associated with autophagy dysregulation, and drugs modulating autophagy have been successful in several animal models. Microglial cells are phagocytes in the central nervous system (CNS) that become activated in pathological conditions and determine the fate of other neural cells. Here, we studied the effects of autophagy on the production of pro-inflammatory molecules in microglial cells and their effects on neuronal cells. We observed that both trehalose and rapamycin activate autophagy in BV2 microglial cells and down-regulate the production of pro-inflammatory cytokines and nitric oxide (NO), in response to LPS and alpha-synuclein. Autophagy also modulated the phosphorylation of p38 and ERK1/2 MAPKs in BV2 cells, which was required for NO production. These actions of autophagy modified the impact of microglial activation on neuronal cells, leading to suppression of neurotoxicity. Our results demonstrate a novel role for autophagy in the regulation of microglial cell activation and pro-inflammatory molecule secretion, which may be important for the control of inflammatory responses in the CNS and neurotoxicity. PMID:28256519

  1. Alpha-Synuclein Expression in the Oligodendrocyte Lineage: an In Vitro and In Vivo Study Using Rodent and Human Models

    Directory of Open Access Journals (Sweden)

    Mehdi Djelloul

    2015-08-01

    Full Text Available In this study, we sought evidence for alpha-synuclein (ASYN expression in oligodendrocytes, as a possible endogenous source of ASYN to explain its presence in glial inclusions found in multiple system atrophy (MSA and Parkinson’s disease (PD. We identified ASYN in oligodendrocyte lineage progenitors isolated from the rodent brain, in oligodendrocytes generated from embryonic stem cells, and in induced pluripotent stem cells produced from fibroblasts of a healthy individual and patients diagnosed with MSA or PD, in cultures in vitro. Notably, we observed a significant decrease in ΑSYN during oligodendrocyte maturation. Additionally, we show the presence of transcripts in PDGFRΑ/CD140a+ cells and SOX10+ oligodendrocyte lineage nuclei isolated by FACS from rodent and human healthy and diseased brains, respectively. Our work identifies ASYN in oligodendrocyte lineage cells, and it offers additional in vitro cellular models that should provide significant insights of the functional implication of ASYN during oligodendrocyte development and disease.

  2. Analysis of striatal transcriptome in mice overexpressing human wild-type alpha-synuclein supports synaptic dysfunction and suggests mechanisms of neuroprotection for striatal neurons

    Directory of Open Access Journals (Sweden)

    Cabeza-Arvelaiz Yofre

    2011-12-01

    Full Text Available Abstract Background Alpha synuclein (SNCA has been linked to neurodegenerative diseases (synucleinopathies that include Parkinson's disease (PD. Although the primary neurodegeneration in PD involves nigrostriatal dopaminergic neurons, more extensive yet regionally selective neurodegeneration is observed in other synucleinopathies. Furthermore, SNCA is ubiquitously expressed in neurons and numerous neuronal systems are dysfunctional in PD. Therefore it is of interest to understand how overexpression of SNCA affects neuronal function in regions not directly targeted for neurodegeneration in PD. Results The present study investigated the consequences of SNCA overexpression on cellular processes and functions in the striatum of mice overexpressing wild-type, human SNCA under the Thy1 promoter (Thy1-aSyn mice by transcriptome analysis. The analysis revealed alterations in multiple biological processes in the striatum of Thy1-aSyn mice, including synaptic plasticity, signaling, transcription, apoptosis, and neurogenesis. Conclusion The results support a key role for SNCA in synaptic function and revealed an apoptotic signature in Thy1-aSyn mice, which together with specific alterations of neuroprotective genes suggest the activation of adaptive compensatory mechanisms that may protect striatal neurons in conditions of neuronal overexpression of SNCA.

  3. shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity.

    Directory of Open Access Journals (Sweden)

    Susana A Gonçalves

    2016-04-01

    Full Text Available Alpha-Synuclein (aSyn misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.

  4. A triple-emission fluorescent probe reveals distinctive amyloid fibrillar polymorphism of wild-type alpha-synuclein and its familial Parkinson's disease mutants.

    Science.gov (United States)

    Celej, M Soledad; Caarls, Wouter; Demchenko, Alexander P; Jovin, Thomas M

    2009-08-11

    Intracytoplasmic neuronal deposits containing amyloid fibrils of the 140-amino acid presynaptic protein alpha-synuclein (AS) are the hallmark of Parkinson's (PD) disease and related neurodegenerative disorders. Three point mutations (A53T, A30P, and E46K) are linked to early onset PD. Compared to the wild-type (WT) protein, the mutants aggregate faster in vitro, but their fibrillar products are quite similar. Using the extrinsic multiple-emission probe 4'-(diethylamino)-3-hydroxyflavone (FE), we demonstrate unique and distinct spectroscopic signatures for the amyloid fibrils formed by the WT and mutant AS, presumably indicative of subtle differences in supramolecular structure. The two well-separated emission bands of the FE probe originate from a proton transfer reaction in the excited state. The ratiometric response constitutes a sensitive, tunable reporter of microenvironmental properties such as polarity and hydrogen bonding. The very distinctive fluorescence spectra of the FE probe bound to the four AS variants reflect different tautomeric equilibria in the excited state and the existence of at least two different binding sites in the fibrils for the dye. Deconvolution of the two-dimensional excitation-emission spectra leads to estimations of different local dielectric constants and extents of hydration characteristic of the proteins. The sensitivity of such a simple external probe to conformational alterations induced by point mutations is unprecedented and provides new insight into key phenomena related to amyloid fibrils: plasticity, polymorphism, propagation of structural features, and structure-function relationships underlying toxicity.

  5. Simultaneous biological removal of endosulfan (alpha+beta) and nitrates from drinking waters using wheat straw as substrate.

    Science.gov (United States)

    Aslan, Sükrü; Türkman, Ayşen

    2004-06-01

    Nitrate and endosulfan (alpha+beta) removal was studied in an upflow biological denitrification reactor packed with wheat straw as carbon source and support particles for microorganisms. While almost complete nitrate elimination and between 65% and 70% endosulfan (alpha+beta) elimination occurred when the temperature was higher than 20 degrees C; below that value, nitrate removal efficiency decreased to about 10%. Nitrate, dissolved organic carbon (DOC), and endosulfan (alpha+beta) removal efficiencies decreased considerably at 1500 microg/l endosulfan concentration in the batch experiments. Although a high removal efficiency was observed for endosulfan (alpha+beta) and nitrate in the biological denitrification continuous reactor, the effluent water could not be used for drinking purpose because of the unacceptable levels of endosulfan (alpha+beta), colour and dissolved organic content. During the continuous study, 23.4% of the initial weight of wheat straw was lost and 24 g was consumed per gram of nitrogen removed. The results of the continuous study showed that 21.3% of the endosulfan removal was achieved by adsorption onto the wheat straw and 68.2% of the endosulfan removal occurred by biological activity and the remaining portion was detected in the effluent water.

  6. Role of N-terminal methionine residues in the redox activity of copper bound to alpha-synuclein.

    Science.gov (United States)

    Rodríguez, Esaú E; Arcos-López, Trinidad; Trujano-Ortiz, Lidia G; Fernández, Claudio O; González, Felipe J; Vela, Alberto; Quintanar, Liliana

    2016-09-01

    Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1-6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1-6) fragment, and in the redox activity of the Cu-AS(1-6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)-AS(1-6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu-AS(1-6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu-AS complex.

  7. The anti-parkinsonian drug selegiline (R(-)-deprenyl) inhibits the nucleation phase of {alpha}-synuclein aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Follmer, Cristian; Braga, Carolina A.; Khattar, Elias; Palhano, Fernando; Freitas, Monica S.; Silva, Jerson L.; Foguel, Debora [Universidade Federal do Rio de Janeiro (UFRJ), RJ (Brazil). Inst. de Bioquimica Medica; Lara, Flavio Alves [Fundacao Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ (Brazil). Lab. de Microbiologia Celular; Lashuel, Hilal [Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland)

    2008-07-01

    Full text: Parkinson's disease (P D) is a chronic disorder characterized by the formation of intra neuronal inclusions called Le wy bodies mainly composed of a-synuclein (a-syn), a natively- unfolded protein with unknown function. Its implication in P D is due to the fact that two mutations (A30P and A53T) are linked to early-onset forms of P D. Selegiline (R(-)-deprenyl) is a noncompetitive monoamino oxidase-B inhibitor which has ne uroprotective effects. It has been administered to P D patients either as monotherapy or in combination with L-dopa. However, its mechanism is unknown. We evaluated the effect of Sel in the in vitro aggregation of A30P either in the presence or absence of amyloid seeds (small fibrils acting as a nucleus). We observed that Sel (1:0.5 or 1:1.5 protein:Sel ratio ) delays fibril formation by enhancing the nucleation phase. Sel effects on fibril formation are abolished when previously added seeds are present, suggesting that Sel interferes with nucleus formation, and is dependent of the A30P:Sel ratio. This inhibitory effect of Sel on the nucleation phase was also evaluated by using another amyloidogenic, natively- unfolded protein, Sup35, but in this case, the effect of Sel was not abolished when Sel was added after the end of the lag phase. We also observed that Sel in combination with dopamine (DA) favors fibril formation. Currently, we are mapping A30P-Sel interaction by NMR. We observed that in the presence of Sel (1:2 p tn:Sel ratio), very little changes occur in the HSQC spectra of the isotopically labeled protein. These results suggest that in the presence of DA, Sel favors the conversion of the toxic prot ofibrils into the non-toxic fibrils, alleviating the dopaminergic neurons from toxic effects. In the non-dopaminergic neurons, Sel would slow down the fibrillation process, probably by forming large spherical aggregates.

  8. Region-specific tauopathy and synucleinopathy in brain of the alpha-synuclein overexpressing mouse model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Masliah Eliezer

    2011-08-01

    Full Text Available Abstract Background α-synuclein [α-Syn]-mediated activation of GSK-3β leading to increases in hyperphosphorylated Tau has been shown by us to occur in striata of Parkinson's diseased [PD] patients and in animal models of PD. In Alzheimer's disease, tauopathy exists in several brain regions; however, the pattern of distribution of tauopathy in other brain regions of PD or in animal models of PD is not known. The current studies were undertaken to analyze the distribution of tauopathy in different brain regions in a widely used mouse model of PD, the α-Syn overexpressing mouse. Results High levels of α-Syn levels were seen in the brain stem, with a much smaller increase in the frontal cortex; neither cerebellum nor hippocampus showed any overexpression of α-Syn. Elevated levels of p-Tau, hyperphosphorylated at Ser202, Ser262 and Ser396/404, were seen in brain stem, with lower levels seen in hippocampus. In both frontal cortex and cerebellum, increases were seen only in p-Ser396/404 Tau, but not in p-Ser202 and p-Ser262. p-GSK-3β levels were not elevated in any of the brain regions, although total GSK-3β was elevated in brain stem. p-p38MAPK levels were unchanged in all brain regions examined, while p-ERK levels were elevated in brain stem, hippocampus and cerebellum, but not the frontal cortex. p-JNK levels were increased in brain stem and cerebellum but not in the frontal cortex or hippocampus. Elevated levels of free tubulin, indicating microtubule destabilization, were seen only in the brain stem. Conclusion Our combined data suggest that in this animal model of PD, tauopathy, along with microtubule destabilization, exists primarily in the brain stem and striatum, which are also the two major brain regions known to express high levels of α-Syn and undergo the highest levels of degeneration in human PD. Thus, tauopathy in PD may have a very restricted pattern of distribution.

  9. Cuminaldehyde as the Major Component of Cuminum cyminum, a Natural Aldehyde with Inhibitory Effect on Alpha-Synuclein Fibrillation and Cytotoxicity.

    Science.gov (United States)

    Morshedi, Dina; Aliakbari, Farhang; Tayaranian-Marvian, Amir; Fassihi, Afshin; Pan-Montojo, Francisco; Pérez-Sánchez, Horacio

    2015-10-01

    Fibrillation of alpha-synuclein (α-SN) is a critical process in the pathophysiology of several neurodegenerative diseases, especially Parkinson's disease. Application of bioactive inhibitory compounds from herbal extracts is a potential therapeutic approach for this cytotoxic process. Here, we investigated the inhibitory effects of the Iranian Cuminum cyminum essential oil on the fibrillation of α-SN. Analysis of different fractions from the total extract identified cuminaldehyde as the active compound involved in the antifibrillation activity. In comparison with baicalein, a well-known inhibitor of α-SN fibrillation, cuminaldehyde showed the same activity in some aspects and a different activity on other parameters influencing α-SN fibrillation. The presence of spermidine, an α-SN fibrillation inducer, dominantly enforced the inhibitory effects of cuminaldehyde even more intensively than baicalein. Furthermore, the results from experiments using preformed fibrils and monobromobimane-labeled monomeric protein also suggest that cuminaldehyde prevents α-SN fibrillation even in the presence of seeds, having no disaggregating impact on the preformed fibrils. Structural studies showed that cuminaldehyde stalls protein assembly into β-structural fibrils, which might be achieved by the interaction with amine groups through its aldehyde group as a Schiff base reaction. This assumption was supported by FITC labeling efficiency assay. In addition, cytotoxicity assays on PC12 cells showed that cuminaldehyde is a nontoxic compound, treatment with cuminaldehyde throughout α-SN fibrillation showed no toxic effects on the cells. Taken together, these results show for the first time that the small abundant natural compound, cuminaldehyde, can modulate α-SN fibrillation. Hence, suggesting that such natural active aldehyde could have potential therapeutic applications.

  10. Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy

    Science.gov (United States)

    Roeters, Steven J.; Iyer, Aditya; Pletikapić, Galja; Kogan, Vladimir; Subramaniam, Vinod; Woutersen, Sander

    2017-01-01

    The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson’s disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fibrils varies as a function of ionic strength: fibrils aggregated in low ionic-strength buffers ([NaCl] ≤ 25 mM) have a significantly different structure than fibrils grown in higher ionic-strength buffers. The observations for fibrils aggregated in low-salt buffers are consistent with an extended conformation of αS molecules, forming hydrogen-bonded intermolecular β-sheets that are loosely packed in a parallel fashion. For fibrils aggregated in high-salt buffers (including those prepared in buffers with a physiological salt concentration) the measurements are consistent with αS molecules in a more tightly-packed, antiparallel intramolecular conformation, and suggest a structure characterized by two twisting stacks of approximately five hydrogen-bonded intermolecular β-sheets each. We find evidence that the high-frequency peak in the amide-I spectrum of αS fibrils involves a normal mode that differs fundamentally from the canonical high-frequency antiparallel β-sheet mode. The high sensitivity of the fibril structure to the ionic strength might form the basis of differences in αS-related pathologies.

  11. The heterozygous A53T mutation in the alpha-synuclein gene in a Chinese Han patient with Parkinson disease: case report and literature review.

    Science.gov (United States)

    Xiong, Wei-Xi; Sun, Yi-Min; Guan, Rong-Yuan; Luo, Su-Shan; Chen, Chen; An, Yu; Wang, Jian; Wu, Jian-Jun

    2016-10-01

    The missense mutation A53T of alpha-synuclein gene (SNCA) was reported to be a rare but definite cause of sporadic and familial Parkinson disease (PD). It seemed to be restricted geographically in Greece and Italy. We aimed to identify the SNCA mutations in a Chinese PD cohort. Ninety-one early onset PD patients or familial PD probands were collected consecutively for the screening of PD-related genes. The genetic analysis was carried out by target sequencing of the exons and the corresponding flanking regions of the PD-related genes using Illumina HiSeq 2000 sequencer and further confirmed by Sanger sequencing or restriction fragment length polymorphism. Dosage mutations of exons in these genes were carried out by multiple ligation-dependent probe amplification. Among the 91 patients, we found only one heterozygous mutation of SNCA A53T, in a 23-year-old male patient with negative family history. The [(11)C]-2β-carbomethoxy-3β-(4-fluorophenyl) tropan (CFT) PET and PD-related spatial covariance pattern (PDRP) via [(18)F]-fluorodeoxyglucos (FDG) PET confirmed a typical pattern of PD. After examining his parents, we found his mother was an asymptomatic carrier, with declined hand dexterity detected by quantitative motor tests. Reduced dopamine transporter uptake of his mother was identified by CFT PET, and abnormal PDRP pattern was found by FDG PET. Our investigation expanded the clinical and genetic spectrum of Chinese PD patients, and we suggested SNCA mutations to be screened in familial and early onset Chinese PD patients.

  12. Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

    Directory of Open Access Journals (Sweden)

    Hélène Hall

    Full Text Available Intraneuronal inclusions containing alpha-synuclein (a-syn constitute one of the pathological hallmarks of Parkinson's disease (PD and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

  13. Chemical Reactivity of alpha-Pinene-derived Products in the Aqueous Phase: Implications on the Fate of Organic Nitrates

    Science.gov (United States)

    Rindelaub, J. D.; Hostetler, M. A.; Lipton, M. A.; Shepson, P. B.

    2014-12-01

    The production of organic nitrates has significant atmospheric importance due to the impact on regional air quality by influencing NOx lifetimes and ozone formation. Additionally, these low volatility compounds readily partition into the particle phase and are important contributors to secondary organic aerosol. Once in the aerosol phase, organic nitrates undergo further chemical reactions that govern their fate in the atmosphere and, consequently, their impact on air quality. Recent research indicates that the presence of water on aerosol particles has a major impact on the reactivity of organic nitrates and that condensed phase hydrolysis leads to the destruction of organic nitrate species, depending on structure. Despite this knowledge, the chemical mechanisms, products, product reactivity and volatility are still uncertain, negatively impacting our understanding of aerosol phase processing and the contribution to air quality. To further understand the atmospheric impact of aerosol phase hydrolysis, we analyzed both condensed phase hydrolysis reactions involving alpha-pinene-derived standards and alpha-pinene photochemical chamber reaction filter samples, using a suite of spectroscopic and mass spectrometric techniques. We were able to measure the pH-dependent hydrolysis rate constants for several types of organic nitrates and identify specific reaction products. The chemistry involved exhibits a strong dependence on pH, providing important mechanistic clues. The results of this study will significantly contribute to our knowledge of aerosol phase chemistry and the impact on regional air quality with respect to the fate of organic nitrate species.

  14. 5-HT2A Receptor Binding in the Frontal Cortex of Parkinson's Disease Patients and Alpha-Synuclein Overexpressing Mice

    DEFF Research Database (Denmark)

    Rasmussen, Nadja Bredo; Olesen, Mikkel Vestergaard; Brudek, Tomasz;

    2016-01-01

    The 5-HT2A receptor is highly involved in aspects of cognition and executive function and seen to be affected in neurodegenerative diseases like Alzheimer's disease and related to the disease pathology. Even though Parkinson's disease (PD) is primarily a motor disorder, reports of impaired...... executive function are also steadily being associated with this disease. Not much is known about the pathophysiology behind this. The aim of this study was thereby twofold: (1) to investigate 5-HT2A receptor binding levels in Parkinson's brains and (2) to investigate whether PD associated pathology, alpha...

  15. Construction and expression of nucleic acid vaccine pVAXl-h-alpha S1-14o coding human alpha-synuclein

    Institute of Scientific and Technical Information of China (English)

    Jiacai Wang; Yingsong Ouyang; Shaojun Wang; Guoguang Peng; Qin Luo; Side Jiang; Faxiang Wang

    2008-01-01

    BACKGROUND: The deposition of α-synuclein (α-syn) aggregates is a neuropathological feature of Parkinson's disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson's disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson's disease.OBJECTIVE: To construct a recombinant eukaryotic expression vector pVAX1 coding human α-syn and to observe its expression level in COS-7 cells.DESIGN AND SETTING: The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University & Chongqing Key Laboratory of Neurology.MATERIALS: The eukaryotic expression plasmid pVAX1, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA.METHODS: The full-length CDS sequence of the human α-syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn I, Xba I and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn I and Xba I simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAXI-hα-S1-14o were transformed into competent E. coli TOP 10 cells and the positive clones were screened and selected using PCR analysis,restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting.MAIN OUTCOME MEASURES: Identification of an eukaryotic expression vector containing the human α-syn gene, pVAX1-hα-S1-140, and detection of the expression in mammalian cell COS-7.RESULTS: The pVAXi vector was successfully cloned with human a -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX1-hα-S1-140 with the human α-syn gene were shown to be expressed in COS-7 cells. Human α-syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western

  16. Selective expression of alpha-synuclein-immunoreactivity in vesicular acetylcholine transporter-immunoreactive axons in the guinea pig rectum and human colon

    NARCIS (Netherlands)

    Sharrad, Dale F.; de Vries, Elsbeth; Brookes, Simon J. H.

    2013-01-01

    Parkinson's disease is a neurodegenerative disorder characterized by motor and nonmotor impairments, including constipation. The hallmark pathological features of Parkinson's disease are Lewy bodies and neurites, of which aggregated a-synuclein is a major constituent. Frequently, Lewy pathology is i

  17. Roles of α-synuclein in Parkinson's Disease%α-突触核蛋白在帕金森病中的作用

    Institute of Scientific and Technical Information of China (English)

    翟骁; 段国礼; 沈下贤; 陈晓

    2012-01-01

    帕金森病(PD)患者的路易小体中存在大量α-突触核蛋白寡聚体.目前认为,寡聚体的形成和增多与氧化应激损伤及氧化修饰息息相关.氧化修饰包括硝化修饰和多巴胺氧化加合.α-突触核蛋白作为分子标志物,可能成为临床治疗帕金森病的新途径.%Alpha-synuclein oligomers are the main composition of the Lewy body in patients with Parkinson's disease , they may be associated with oxidative stress damage and oxidative modification. The oxidative modifications include nitrative modification and dopamine oxidative adduction. Serving as a biomarker, a-synuclein may become a new target for clinical treatment.

  18. Perivascular nerve fiber α-synuclein regulates contractility of mouse aorta: a link to autonomic dysfunction in Parkinson's disease.

    Science.gov (United States)

    Marrachelli, Vannina G; Miranda, Francisco J; Alabadí, José A; Milán, Miguel; Cano-Jaimez, Marifé; Kirstein, Martina; Alborch, Enrique; Fariñas, Isabel; Pérez-Sánchez, Francisco

    2010-07-01

    Parkinson's disease and other neurodegenerative disorders associated to changes in alpha-synuclein often result in autonomic dysfunction, most of the time accompanied by abundant expression of this synaptic protein in peripheral autonomic neurons. Given that expression of alpha-synuclein in vascular elements has been previously reported, the present study was undertaken to determine whether alpha-synuclein directly participates in the regulation of vascular responsiveness. We detected by immunohistochemistry perivascular nerve fibers containing alpha-synuclein in the aorta of mice while aortic endothelial cells and muscular fibers themselves did not exhibit detectable levels of this protein. To assess the effect of alpha-synuclein on vascular reactivity, aortic ring preparations obtained from alpha-synuclein-deficient knockout mice and from transgenic mice overexpressing human wild-type alpha-synuclein under the control of the tyrosine hydroxylase-promoter were mounted and equilibrated in organ baths for isometric tension recording. Lack of alpha-synuclein did not modify the relaxant responses to the endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) vasodilators, but resulted in a greater than normal norepinephrine-induced vasoconstriction along with a lowered response to dopamine, suggesting potential presynaptic changes in dopamine and norepinephrine releases in knockout mice. Overexpression of alpha-synuclein in TH-positive fibers resulted in complex abnormal responses, characterized by lowered acetylcholine-induced relaxation and lowered norepinephrin-induced contraction. Taken together, our data show for the first time that alpha-synuclein is present in sympathetic fibers supplying the murine aorta and provide evidence that changes in alpha-synuclein levels in perivascular fibers play a physiological role in the regulation of vascular function.

  19. Candidate inflammatory biomarkers display unique relationships with alpha-synuclein and correlate with measures of disease severity in subjects with Parkinson's disease.

    Science.gov (United States)

    Eidson, Lori N; Kannarkat, George T; Barnum, Christopher J; Chang, Jianjun; Chung, Jaegwon; Caspell-Garcia, Chelsea; Taylor, Peggy; Mollenhauer, Brit; Schlossmacher, Michael G; Ereshefsky, Larry; Yen, Mark; Kopil, Catherine; Frasier, Mark; Marek, Kenneth; Hertzberg, Vicki S; Tansey, Malú G

    2017-08-18

    Efforts to identify fluid biomarkers of Parkinson's disease (PD) have intensified in the last decade. As the role of inflammation in PD pathophysiology becomes increasingly recognized, investigators aim to define inflammatory signatures to help elucidate underlying mechanisms of disease pathogenesis and aid in identification of patients with inflammatory endophenotypes that could benefit from immunomodulatory interventions. However, discordant results in the literature and a lack of information regarding the stability of inflammatory factors over a 24-h period have hampered progress. Here, we measured inflammatory proteins in serum and CSF of a small cohort of PD (n = 12) and age-matched healthy control (HC) subjects (n = 6) at 11 time points across 24 h to (1) identify potential diurnal variation, (2) reveal differences in PD vs HC, and (3) to correlate with CSF levels of amyloid β (Aβ) and α-synuclein in an effort to generate data-driven hypotheses regarding candidate biomarkers of PD. Despite significant variability in other factors, a repeated measures two-way analysis of variance by time and disease state for each analyte revealed that serum IFNγ, TNF, and neutrophil gelatinase-associated lipocalin (NGAL) were stable across 24 h and different between HC and PD. Regression analysis revealed that C-reactive protein (CRP) was the only factor with a strong linear relationship between CSF and serum. PD and HC subjects showed significantly different relationships between CSF Aβ proteins and α-synuclein and specific inflammatory factors, and CSF IFNγ and serum IL-8 positively correlated with clinical measures of PD. Finally, linear discriminant analysis revealed that serum TNF and CSF α-synuclein discriminated between PD and HC with a minimum of 82% sensitivity and 83% specificity. Our findings identify a panel of inflammatory factors in serum and CSF that can be reliably measured, distinguish between PD and HC, and monitor inflammation as disease

  20. Acyl-CoA synthetase activity links wild-type but not mutant a-Synuclein to brain arachidonate metabolism

    DEFF Research Database (Denmark)

    Golovko, Mikhail; Rosenberger, Thad; Færgeman, Nils J.

    2006-01-01

    Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of alpha-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-(14)C]20:4n-6 incorporation and turnover kinetics in brain phospholipids usi...

  1. Evaluation of the effects of vitamin A supplementation on adult rat substantia nigra and striatum redox and bioenergetic states: mitochondrial impairment, increased 3-nitrotyrosine and alpha-synuclein, but decreased D2 receptor contents.

    Science.gov (United States)

    de Oliveira, Marcos Roberto; Oliveira, Max William Soares; Behr, Guilherme Antônio; Hoff, Mariana Leivas Muller; da Rocha, Ricardo Fagundes; Moreira, José Cláudio Fonseca

    2009-03-17

    Vitamin A at moderate to high doses is applied in the treatment of some life threatening pathological conditions, for instance cancers. Additionally, vitamin A at low concentrations is a known antioxidant molecule. However, by increasing vitamin A (or its derivatives) concentrations, there is an increase in the levels of oxidative stress markers in several experimental models. Furthermore, it was reported that vitamin A therapy at high doses might induce cognitive decline among the patients, which may become anxious or depressive, for example, depending on vitamin A levels intake. We have previously reported increased levels of oxidative stress markers in rat substantia nigra and striatum. However, the mechanism by which this vitamin altered the redox environment in such rat brain regions remains to be elucidated. In the herein presented work, we have investigated the effects of vitamin A supplementation at clinical doses (1000-9000 IU/kg day(-1)) for 28 days on rat substantia nigra and striatum mitochondrial electron transfer chain (METC) activity, which may produce superoxide anion radical (O(2)(-*)) when impaired. Additionally, the levels of non-enzymatic antioxidant defenses were evaluated, as well as 3-nitrotyrosine, alpha- and beta-synucleins and TNF-alpha levels through ELISA assay. We observed impaired METC in both rat brain regions. Moreover, we found increased O(2)(-*) production and nitrotyrosine content in the nigrostriatal axis of vitamin A-treated rats, suggesting that the use of vitamin A at therapeutic doses may be rethought due to this toxic effects found here.

  2. Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.

    Directory of Open Access Journals (Sweden)

    Luisel R Lemkau

    Full Text Available Parkinson's disease (PD is pathologically characterized by the presence of Lewy bodies (LBs in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS, a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils.

  3. Alpha-synuclein in the cerebrospinal fluid differentiates synucleinopathies (Parkinson Disease, dementia with Lewy bodies, multiple system atrophy) from Alzheimer disease.

    Science.gov (United States)

    Tateno, Fuyuki; Sakakibara, Ryuji; Kawai, Takayuki; Kishi, Masahiko; Murano, Takeyoshi

    2012-01-01

    We examined the utility of quantification of α-synuclein (SNCA) in the cerebrospinal fluid (CSF) to differentiate patients with Alzheimer disease (AD), dementia with Lewy bodies (DLB), Parkinson disease (PD), and multiple system atrophy (MSA). Thirty-seven patients were divided into 4 age-matched and sex-matched clinical groups: AD (n = 9), DLB (n = 6), PD (n = 11), and MSA (n = 11). Eleven subjects served as neurological disease controls. The total of 48 subjects included 27 men and 21 women, aged 66.5 ± 11.4 years. We performed a solid-phase sandwich enzyme-linked immunosorbent assay, which enables the sensitive quantification of CSF SNCA. In comparison with controls, CSF SNCA levels in AD were significantly higher (P < 0.05). CSF SNCA levels in PD (P < 0.001), DLB (P < 0.01), and MSA (P < 0.05) were all significantly lower than those in AD. However, CSF SNCA levels did not differ significantly among the 3 synucleinopathies. The results of the present study suggest that quantification of CSF SNCA helps in the differentiation of synucleinopathies (PD, DLB, and MSA) from AD. However, CSF SNCA levels did not differ significantly among the 3 synucleinopathies.

  4. The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons.

    Science.gov (United States)

    Salganik, Maxim; Sergeyev, Valeriy G; Shinde, Vishal; Meyers, Craig A; Gorbatyuk, Marina S; Lin, Jonathan H; Zolotukhin, Sergey; Gorbatyuk, Oleg S

    2015-06-01

    Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the endoplasmic reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work, we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose-regulated protein 78 (GRP78) could be related to the development of Parkinson's disease. We first determined that old (24 months) rats exhibit significantly lower levels of GRP78 protein in the nigrostriatal system as compared with young (2 months) animals. Then using recombinant adeno-associate virus-mediated gene transfer, we found that GRP78 downregulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α-syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of 9 months protected aging nigral DA neurons in the α-syn-induced rat model of Parkinson's-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration.

  5. Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study.

    Science.gov (United States)

    Curtain, Cyril C; Kirby, Nigel M; Mertens, Haydyn D T; Barnham, Kevin J; Knott, Robert B; Masters, Colin L; Cappai, Roberto; Rekas, Agata; Kenche, Vijaya B; Ryan, Timothy

    2015-01-01

    The 140 residue intrinsically disordered protein α-synuclein (α-syn) self-associates to form fibrils that are the major constituent of the Lewy body intracellular protein inclusions, and neurotoxic oligomers. Both of these macromolecular structures are associated with a number of neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies. Using ensemble optimisation modelling (EOM) and small angle X-ray scattering (SAXS) on a size-exclusion column equipped beamline, we studied how the distribution of structural conformers in α-syn may be influenced by the presence of the familial early-onset mutations A30P, E45K and A53T, by substituting the four methionine residues with alanines and by reaction with copper (Cu2+) or an anti-fibril organic platinum (Pt) complex. We found that the WT had two major conformer groups, representing ensembles of compact and extended structures. The population of the extended group was increased in the more rapidly fibril-forming E45K and A53T mutants, while the compact group was enlarged in the oligomer-forming A30P mutant. Addition of Cu2+ resulted in the formation of an ensemble of compact conformers, while the anti-fibril agent and alanine substitution substantially reduced the population of extended conformers. Since our observations with the mutants suggest that fibrils may be drawn from the extended conformer ensemble, we propose that the compact and extended ensembles represent the beginning of oligomer and fibril formation pathways respectively, both of which have been reported to lead to a toxic gain of function. Manipulating these pathways and monitoring the results by EOM and SAXS may be useful in the development of anti-Parkinson's disease therapies.

  6. Suppression of MAPK attenuates neuronal cell death induced by activated glia-conditioned medium in alpha-synuclein overexpressing SH-SY5Y cells.

    Science.gov (United States)

    Yshii, Lidia M; Denadai-Souza, Alexandre; Vasconcelos, Andrea R; Avellar, Maria Christina W; Scavone, Cristoforo

    2015-10-26

    Parkinson's disease (PD) is a neurodegenerative disease with characteristics and symptoms that are well defined. Nevertheless, its aetiology remains unknown. PD is characterized by the presence of Lewy bodies inside neurons. α-Synuclein (α-syn) is a soluble protein present in the pre-synaptic terminal of neurons. Evidence suggests that α-syn has a fundamental role in PD pathogenesis, given that it is an important component of Lewy bodies localized in the dopaminergic neurons of PD patients. In the present study, we investigated the influence of wild type (WT) and A30P α-syn overexpression on neuroblastoma SH-SY5Y toxicity induced by the conditioned medium (CM) from primary cultures of glia challenged with lipopolysaccharide (LPS) from Escherichia coli. We observed that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show evidence of cell death, which is not reverted by NF-κB inhibition by sodium salicylate or by blockage of P50 (NF-κB subunit). Furthermore, the expression of A30P α-syn in neuroblastoma SH-SY5Y decreases the cell death triggered by the CM of activated glia versus WT α-syn or control group. This effect of A30P α-syn may be due to the low MAPK42/44 phosphorylation. This finding is substantiated by MEK1 inhibition by PD98059, decreasing LDH release by CM in SH-SY5Y cells. Our results suggest that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show cell death, which is not reverted by NF-κB blockage. Additionally, the expression of A30P α-syn on neuroblastoma SH-SY5Y leads to decreased cell death triggered by the CM of activated glia, when compared to WT α-syn or control group. The mechanism underlying this process remains to be completely elucidated, but the present data suggest that MAPK42/44 phosphorylation plays an important role in this process. CRD42015020829.

  7. The G209A mutation in the alpha-synuclein gene in Brazilian families with Parkinson's disease Mutação G209A no gene da alfa-sinucleína em famílias brasileiras com doença de Parkinson

    Directory of Open Access Journals (Sweden)

    Hélio A.G. Teive

    2001-09-01

    Full Text Available A missense G209A mutation of the alpha-synuclein gene was recently described in a large Contursi kindred with Parkinson's disease (PD. The objective of this study is to determine if the mutation G209A of the alpha-synuclein gene was present in 10 Brazilian families with PD. PD patients were recruited from movement disorders clinics of Brazil. A family history with two or more affected in relatives was the inclusion criterion for this study. The alpha-synuclein G209A mutation assay was made using polymerase chain reaction and the restriction enzyme Tsp45I. Ten patients from 10 unrelated families were studied. The mean age of PD onset was 42.7 years old. We did not find the G209A mutation in our 10 families with PD. Our results suggest that alpha-synuclein mutation G209A is uncommon in Brazilian PD families.Recentemente foi detectada mutação missense G209A no gene da alfa-sinucleína em uma grande família com doença de Parkinson (DP de Contursi, Itália. Este estudo tem o objetivo de determinar se a mutação G209A está presente em 10 famílias brasileiras com DP. Pacientes com DP foram recrutados em clínicas de distúrbio do movimento no Brasil. O critério de inclusão no estudo foi à presença de dois ou mais familiares acometidos pela DP. A mutação G209A do gene da alfa-sinucleína foi pesquisada usando a técnica de reação em cadeia de polimerase e a enzima de restrição Tsp45I. Foram estudados 10 pacientes de famílias não-relacionadas. A idade média do início dos sintomas da DP foi 42,7 anos. Não encontramos a mutação estudada neste grupo de pacientes. Nossos resultados sugerem que a mutação G209A é incomum em famílias brasileiras com DP.

  8. The relation between α-synuclein and microglia in Parkinson's disease: Recent developments.

    Science.gov (United States)

    Sanchez-Guajardo, V; Tentillier, N; Romero-Ramos, M

    2015-08-27

    Recent research suggests a complex role for microglia not only in Parkinson's disease but in other disorders involving alpha-synuclein aggregation, such as multiple system atrophy. In these neurodegenerative processes, the activation of microglia is a common pathological finding, which disturbs the homeostasis of the neuronal environment otherwise maintained, among others, by microglia. The term activation comprises any deviation from what otherwise is considered normal microglia status, including cellular abundance, morphology or protein expression. The microglial response during disease will sustain survival or otherwise promote cell degeneration. The novel concepts of alpha-synuclein being released and uptaken by neighboring cells, and their importance in disease progression, positions microglia as the main cell that can clear and handle alpha-synuclein efficiently. Microglia's behavior will therefore be a determinant on the disease's progression. For this reason we believe that the better understanding of microglia's response to alpha-synuclein pathological accumulation across brain areas and disease stages is essential to develop novel therapeutic tools for Parkinson's disease and other alpha-synucleinopathies. In this review we will revise the most recent findings and developments with regard to alpha-synuclein and microglia in Parkinson's disease.

  9. Redistribution of DAT/α-Synuclein Complexes Visualized by “In Situ” Proximity Ligation Assay in Transgenic Mice Modelling Early Parkinson's Disease

    OpenAIRE

    Arianna Bellucci; Laura Navarria; Elisa Falarti; Michela Zaltieri; Federica Bono; Ginetta Collo; Maria Grazia Spillantini; Cristina Missale; Pierfranco Spano

    2011-01-01

    Alpha-synuclein, the major component of Lewy bodies, is thought to play a central role in the onset of synaptic dysfunctions in Parkinson's disease (PD). In particular, α-synuclein may affect dopaminergic neuron function as it interacts with a key protein modulating dopamine (DA) content at the synapse: the DA transporter (DAT). Indeed, recent evidence from our "in vitro" studies showed that α-synuclein aggregation decreases the expression and membrane trafficking of the DAT as the DAT is ret...

  10. Highly potent and specific GSK-3beta inhibitors that block tau phosphorylation and decrease alpha-synuclein protein expression in a cellular model of Parkinson's disease.

    Science.gov (United States)

    Kozikowski, Alan P; Gaisina, Irina N; Petukhov, Pavel A; Sridhar, Jayalakshmi; King, LaShaunda T; Blond, Sylvie Y; Duka, Tetyana; Rusnak, Milan; Sidhu, Anita

    2006-02-01

    Research by Klein and co-workers suggests that the inhibition of GSK-3beta by small molecules may offer an important strategy in the treatment of a number of central nervous system (CNS) disorders including Alzheimer's disease, Parkinson's disease, and bipolar disorders. Based on results from kinase-screening assays that identified a staurosporine analogue as a modest inhibitor of GSK-3beta, a series of 3-indolyl-4-indazolylmaleimides was prepared for study in both enzymatic and cell-based assays. Most strikingly, whereas we identified ligands having poor to high potency for GSK-3beta inhibition, only ligands with a Ki value of less than 8 nM, namely maleimides 18 and 22, were found to inhibit Tau phosphorylation at a GSK-3beta-specific site (Ser 396/404). Accordingly, maleimides 18 and 22 may protect neuronal cells against cell death by decreasing the level of alpha-Syn protein expression. We conclude that the GSK-3beta inhibitors described herein offer promise in defending cells against MPP+-induced neurotoxicity and that such compounds will be valuable to explore in animal models of Parkinson's disease as well as in other Tau-related neurodegenerative disease states.

  11. α-Synuclein posttranslational modification and alternative splicing as a trigger for neurodegeneration.

    Science.gov (United States)

    Beyer, Katrin; Ariza, Aurelio

    2013-04-01

    Lewy body diseases include Parkinson disease and dementia with Lewy bodies and are characterized by the widespread distribution of Lewy bodies in virtually every brain area. The main component of Lewy bodies is alpha-synuclein (AS). Accumulating evidence suggests that AS oligomerization and aggregation are strongly associated with the pathogenesis of Lewy body diseases. AS is a small soluble protein with aggregation-prone properties under certain conditions. These properties are enhanced by posttranslational modifications such as phosphorylation, ubiquitination, nitration, and truncation. Accordingly, Lewy bodies contain abundant phosphorylated, nitrated, and monoubiquitinated AS. However, alternative splicing of the AS gene is also known to modify AS aggregation propensities. Splicing gives rise to four related forms of the protein, the main transcript and those that lack exon 4, exon 6, or both. Since AS structure and properties have been extensively studied, it is possible to predict the consequences of the splicing out of the two aforesaid exons. The present review discusses the latest insights on the mechanisms of AS posttranslational modifications and intends to depict their role in the pathogenesis of Lewy body diseases. The implications of deregulated alternative splicing are examined as well, and a hypothesis for the development of the pure form of dementia with Lewy bodies is proposed.

  12. Assays for alpha-synuclein aggregation

    DEFF Research Database (Denmark)

    Giehm, Lise; Lorenzen, Nikolai; Otzen, Daniel

    2011-01-01

    leading to their formation is important for designing new drugs as well as in development of new nano-biomaterials such as nano-tubes, wires, scaffolds etc. 6. Understanding the process of amyloid formation requires an ability to reproduce this aggregation under controlled circumstances, in other words...

  13. Reversal of effects of intra peritoneally administered beryllium nitrate by tiron and CaNa3DTPA alone or in combination with alpha-tocopherol.

    Science.gov (United States)

    Nirala, Satendra Kumar; Bhadauria, Monika; Upadhyay, Anil Kumar; Mathur, Ramesh; Mathur, Asha

    2009-12-01

    To evaluate therapeutic efficacy of chelating agents tiron (Sodium-4,5-dihydroxy-1,3-benzene disulphonate) and CaNa3DTPA (Calcium trisodium diethylene triamine pentaacetic acid) in presence of alpha-tocopherol against beryllium induced toxicity, adult female albino rats were exposed to beryllium nitrate for 28 days followed by therapy with tiron (471 mg/kg, i.p.) and CaNa3DTPA (35 mg/kg, i.p.) alone and in combination with alpha-tocopherol (25 mg/kg, p.o.). Results revealed non-significant fall in haemoglobin and total serum protein content while significant fall in blood sugar level and activity of serum alkaline phosphatase. On the other hand, significant rise in the activity of serum transaminases and LDH was noticed after beryllium administration. Significant increase in total and esterified cholesterol was found in liver and kidney after toxicity. Significant increase in lipid peroxidation and decreased level of reduced glutathione in both the organs showed oxidative stress due to beryllium exposure. Histopathological and ultrastructural observations of liver and kidney revealed lesions due to beryllium toxicity followed by recovery due to combined therapy. CaNa3DTPA showed moderate therapeutic efficacy; however, its effectiveness was enhanced with alpha-tocopherol to some extent. Tiron in combination with alpha-tocopherol exerted statistically more beneficial effects in reversal of beryllium induced biochemical, histopathological and ultrastructural alterations.

  14. Fish Synucleins: An Update

    Directory of Open Access Journals (Sweden)

    Mattia Toni

    2015-10-01

    Full Text Available Synucleins (syns are a family of proteins involved in several human neurodegenerative diseases and tumors. Since the first syn discovery in the brain of the electric ray Torpedo californica, members of the same family have been identified in all vertebrates and comparative studies have indicated that syn proteins are evolutionary conserved. No counterparts of syns were found in invertebrates suggesting that they are vertebrate-specific proteins. Molecular studies showed that the number of syn members varies among vertebrates. Three genes encode for α-, β- and γ-syn in mammals and birds. However, a variable number of syn genes and encoded proteins is expressed or predicted in fish depending on the species. Among biologically verified sequences, four syn genes were identified in fugu, encoding for α, β and two γ (γ1 and γ2 isoforms, whereas only three genes are expressed in zebrafish, which lacks α-syn gene. The list of “non verified” sequences is much longer and is often found in sequence databases. In this review we provide an overview of published papers and known syn sequences in agnathans and fish that are likely to impact future studies in this field. Indeed, fish models may play a key role in elucidating some of the molecular mechanisms involved in physiological and pathological functions of syn proteins.

  15. THE EFFECT OF GESTATIONAL MERCURY VAPOR EXPOSURE ON RAT BRAIN A-SYNUCLEIN EXPRESSION.

    Science.gov (United States)

    Alpha-synuclein is a highly conserved protein that localizes to pre-synaptic terminals and is thought to play a role in neuronal plasticity. It is upregulated developmentally and continues to be expressed at high levels in the adult brain. Its presence in a number of neuronal (A...

  16. Synucleins regulate the kinetics of synaptic vesicle endocytosis.

    Science.gov (United States)

    Vargas, Karina J; Makani, Sachin; Davis, Taylor; Westphal, Christopher H; Castillo, Pablo E; Chandra, Sreeganga S

    2014-07-09

    Genetic and pathological studies link α-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. α-Synuclein, an acidic lipid binding protein, shares high sequence identity with β- and γ-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse α-, β-, or γ-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knock-out synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize α-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD.

  17. Effects of Human Alpha-Synuclein A53T-A30P Mutations on SVZ and Local Olfactory Bulb Cell Proliferation in a Transgenic Rat Model of Parkinson Disease

    Directory of Open Access Journals (Sweden)

    Faustine Lelan

    2011-01-01

    Full Text Available A transgenic Sprague Dawley rat bearing the A30P and A53T α-synuclein (α-syn human mutations under the control of the tyrosine hydroxylase promoter was generated in order to get a better understanding of the role of the human α-syn mutations on the neuropathological events involved in the progression of the Parkinson’s disease (PD. This rat displayed olfactory deficits in the absence of motor impairments as observed in most early PD cases. In order to investigate the role of the mutated α-syn on cell proliferation, we focused on the subventricular zone (SVZ and the olfactory bulbs (OB as a change of the proliferation could affect OB function. The effect on OB dopaminergic innervation was investigated. The human α-syn co-localized in TH-positive OB neurons. No human α-syn was visualized in the SVZ. A significant increase in resident cell proliferation in the glomerular but not in the granular layers of the OB and in the SVZ was observed. TH innervation was significantly increased within the glomerular layer without an increase in the size of the glomeruli. Our rat could be a good model to investigate the role of human mutated α-syn on the development of olfactory deficits.

  18. A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3′end using CRISPR-Cas9 genome editing technique

    Science.gov (United States)

    Basu, Sambuddha; Adams, Levi; Guhathakurta, Subhrangshu; Kim, Yoon-Seong

    2017-01-01

    α-synuclein (α-SYN) is a major pathologic contributor to Parkinson’s disease (PD). Multiplication of α-SYN encoding gene (SNCA) is correlated with early onset of the disease underlining the significance of its transcriptional regulation. Thus, monitoring endogenous transcription of SNCA is of utmost importance to understand PD pathology. We developed a stable cell line expressing α-SYN endogenously tagged with NanoLuc luciferase reporter using CRISPR/Cas9-mediated genome editing. This allows efficient measurement of transcriptional activity of α-SYN in its native epigenetic landscape which is not achievable using exogenous transfection-based luciferase reporter assays. The NanoLuc activity faithfully monitored the transcriptional regulation of SNCA following treatment with different drugs known to regulate α-SYN expression; while exogenous promoter-reporter assays failed to reproduce the similar outcomes. To our knowledge, this is the first report showing endogenous monitoring of α-SYN transcription, thus making it an efficient drug screening tool that can be used for therapeutic intervention in PD. PMID:28374838

  19. Resistance to MPTP-neurotoxicity in α-synuclein knockout mice is complemented by human α-synuclein and associated with increased β-synuclein and Akt activation.

    Directory of Open Access Journals (Sweden)

    Bobby Thomas

    Full Text Available Genetic and biochemical abnormalities of α-synuclein are associated with the pathogenesis of Parkinson's disease. In the present study we investigated the in vivo interaction of mouse and human α-synuclein with the potent parkinsonian neurotoxin, MPTP. We find that while lack of mouse α-synuclein in mice is associated with reduced vulnerability to MPTP, increased levels of human α-synuclein expression is not associated with obvious changes in the vulnerability of dopaminergic neurons to MPTP. However, expressing human α-synuclein variants (human wild type or A53T in the α-synuclein null mice completely restores the vulnerability of nigral dopaminergic neurons to MPTP. These results indicate that human α-synuclein can functionally replace mouse α-synuclein in regard to vulnerability of dopaminergic neurons to MPTP-toxicity. Significantly, α-synuclein null mice and wild type mice were equally sensitive to neurodegeneration induced by 2'NH(2-MPTP, a MPTP analog that is selective for serotoninergic and noradrenergic neurons. These results suggest that effects of α-synuclein on MPTP like compounds are selective for nigral dopaminergic neurons. Immunoblot analysis of β-synuclein and Akt levels in the mice reveals selective increases in β-synuclein and phosphorylated Akt levels in ventral midbrain, but not in other brain regions, of α-synuclein null mice, implicating the α-synuclein-level dependent regulation of β-synuclein expression in modulation of MPTP-toxicity by α-synuclein. Together these findings provide new mechanistic insights on the role α-synuclein in modulating neurodegenerative phenotypes by regulation of Akt-mediated cell survival signaling in vivo.

  20. β-Synuclein suppresses both the initiation and amplification steps of α-synuclein aggregation via competitive binding to surfaces

    Science.gov (United States)

    Brown, James W. P.; Buell, Alexander K.; Michaels, Thomas C. T.; Meisl, Georg; Carozza, Jacqueline; Flagmeier, Patrick; Vendruscolo, Michele; Knowles, Tuomas P. J.; Dobson, Christopher M.; Galvagnion, Céline

    2016-11-01

    α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson’s disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.

  1. Nitration of Naphthol: A Laboratory Experiment.

    Science.gov (United States)

    Mowery, Dwight F.

    1982-01-01

    Products of nitrations, upon distillation or steam distillation, may produce dermatitis in some students. A procedure for nitration of beta-naphthol producing a relatively non-volatile product not purified by steam distillation is described. Nitration of alpha-naphthol by the same procedure yields Martius Yellow dye which dyes wool yellow or…

  2. Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Sun Xuan

    2010-10-01

    Full Text Available Abstract Background The pathological hallmarks of Parkinson's disease (PD include the presence of alpha-synuclein (α-syn rich Lewy bodies and neurites and the loss of dopaminergic (DA neurons of the substantia nigra (SN. Animal models of PD based on viral vector-mediated over-expression of α-syn have been developed and show evidence of DA toxicity to varying degrees depending on the type of virus used, its concentration, and the serotype of vector employed. To date these models have been variable, difficult to reproduce, and slow in their evolution to achieve a desired phenotype, hindering their use as a model for testing novel therapeutics. To address these issues we have taken a novel vector in this context, that can be prepared in high titer and which possesses an ability to produce neuronally-directed expression, with expression dynamics optimised to provide a rapid rise in gene product expression. Thus, in the current study, we have used a high titer chimeric AAV1/2 vector, to express human A53T α-syn, an empty vector control (EV, or green fluorescent protein (GFP, the latter to control for the possibility that high levels of protein in themselves might contribute to damage. Results We show that following a single 2 μl injection into the rat SN there is near complete coverage of the structure and expression of A53T α-syn or GFP appears throughout the striatum. Within 3 weeks of SN delivery of their respective vectors, aggregations of insoluble α-syn were observed in SN DA neurons. The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%, and to a lesser extent by GFP (24%, compared to EV controls (both P P Conclusions In the current implementation of the model, we recapitulate the primary pathological hallmarks of PD, although a proportion of the SN damage may relate to general protein overload and may not be specific for A53T α-syn. Future studies will thus be required to optimise the dose of

  3. Phosphorylated α-synuclein in Parkinson's disease

    DEFF Research Database (Denmark)

    Stewart, Tessandra; Sossi, Vesna; Aasly, Jan O;

    2015-01-01

    INTRODUCTION: α-Synuclein (α-syn) is a key protein in Parkinson's disease (PD), and one of its phosphorylated forms, pS129, is higher in PD patients than healthy controls. However, few studies have examined its levels in longitudinally collected cerebrospinal fluid (CSF) or in preclinical cases. ...

  4. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... storage in the leaf vacuole cells to be released later and reduced in the cytosol ... pathway dependent on nitrate ion concentration, and (2) potassium and ..... converted to starch in storage organs (Li et al., 2009;. Amtmann and ...

  5. p25alpha relocalizes in oligodendroglia from myelin to cytoplasmic inclusions in multiple system atrophy

    DEFF Research Database (Denmark)

    Song, Yun Ju C; Lundvig, Ditte M S; Huang, Yue

    2007-01-01

    was revealed by immunoblotting along with the presence of immunoreactivity for MBP degradation products in oligodendroglia. The oligodendroglial cell bodies in MSA displayed an enlargement along with the relocalization of p25alpha, and this was enhanced after the deposition of alpha-synuclein in the glial...... cytoplasmic inclusions. Overall, the data indicate that changes in the cellular interactions between MBP and p25alpha occur early in MSA and contribute to abnormalities in myelin and subsequent alpha-synuclein aggregation and the ensuing neuronal degeneration that characterizes this disease....

  6. Mechanisms of alpha-Synuclein Aggregation and Toxicity

    Science.gov (United States)

    2006-09-01

    K. R., Krajcarski, D. T., and Rayner, D. M. (1978) FEBS Lett. 94, 249–252 31. Thomson, A. M., Rogers , J. T., and Leedman, P. J. (1999) Int. J...McNaught, K.S., and Jenner, P. (2001). Proteasomal function is im- Chartier -Harlin, M. (2000). Linkage exclusion in French families with paired in

  7. Alpha-Synuclein Oligomers: an Amyloid Pore? Insights into Mechanisms of alpha-Synuclein Oligomer-Lipid Interactions

    NARCIS (Netherlands)

    Stockl, Martin T.; Zijlstra, Niels; Subramaniam, Vinod

    2013-01-01

    In many human diseases, oligomeric species of amyloid proteins may play a pivotal role in cytotoxicity. Many lines of evidence indicate that permeabilization of cellular membranes by amyloid oligomers may be the key factor in disrupting cellular homeostasis. However, the exact mechanisms by which th

  8. alpha-Synuclein Oligomers: an Amyloid Pore? : Insights into Mechanisms of alpha-Synuclein Oligomer-Lipid Interactions

    NARCIS (Netherlands)

    Stockl, M.T.; Zijlstra, N.; Subramaniam, V.

    2013-01-01

    In many human diseases, oligomeric species of amyloid proteins may play a pivotal role in cytotoxicity. Many lines of evidence indicate that permeabilization of cellular membranes by amyloid oligomers may be the key factor in disrupting cellular homeostasis. However, the exact mechanisms by which th

  9. Potential Modes of Intercellular α-Synuclein Transmission

    Directory of Open Access Journals (Sweden)

    Dario Valdinocci

    2017-02-01

    Full Text Available Intracellular aggregates of the α-synuclein protein result in cell loss and dysfunction in Parkinson’s disease and atypical Parkinsonism, such as multiple system atrophy and dementia with Lewy bodies. Each of these neurodegenerative conditions, known collectively as α-synucleinopathies, may be characterized by a different suite of molecular triggers that initiate pathogenesis. The mechanisms whereby α-synuclein aggregates mediate cytotoxicity also remain to be fully elucidated. However, recent studies have implicated the cell-to-cell spread of α-synuclein as the major mode of disease propagation between brain regions during disease progression. Here, we review the current evidence for different modes of α-synuclein cellular release, movement and uptake, including exocytosis, exosomes, tunneling nanotubes, glymphatic flow and endocytosis. A more detailed understanding of the major modes by which α-synuclein pathology spreads throughout the brain may provide new targets for therapies that halt the progression of disease.

  10. Low CSF levels of both α-synuclein and the α-synuclein cleaving enzyme neurosin in patients with synucleinopathy.

    Directory of Open Access Journals (Sweden)

    Malin Wennström

    Full Text Available Neurosin is a protease that in vitro degrades α-synuclein, the main constituent of Lewy bodies found in brains of patients with synucleinopathy including Parkinson's disease (PD and dementia with Lewy bodies (DLB. Several studies have reported reduced cerebrospinal fluid (CSF levels of α-synuclein in synucleinopathy patients and recent data also proposes a significant role of α-synuclein in the pathophysiology of Alzheimer's disease (AD. To investigate potential links between neurosin and its substrate α-synuclein in vivo we used a commercially available sandwich ELISA and an in-house developed direct ELISA to quantify CSF levels of α-synuclein and neurosin in patients diagnosed with DLB, PD and PD dementia (PDD versus AD patients and non-demented controls. We found that patients with synucleinopathy displayed lower CSF levels of neurosin and α-synuclein compared to controls and AD patients. In contrast, AD patients demonstrated significantly increased CSF α-synuclein but similar neurosin levels compared to non-demented controls. Further, CSF neurosin and α-synuclein concentrations were positively associated in controls, PD and PDD patients and both proteins were highly correlated to CSF levels of phosphorylated tau in all investigated groups. We observed no effect of gender or presence of the apolipoprotein Eε4 allele on neither neurosin or α-synuclein CSF levels. In concordance with the current literature our study demonstrates decreased CSF levels of α-synuclein in synucleinopathy patients versus AD patients and controls. Importantly, decreased α-synuclein levels in patients with synucleinopathy appear linked to low levels of the α-synuclein cleaving enzyme neurosin. In contrast, elevated levels of α-synuclein in AD patients were not related to any altered CSF neurosin levels. Thus, altered CSF levels of α-synuclein and neurosin in patients with synucleinopathy versus AD may not only mirror disease-specific neuropathological

  11. Insensitive Ammonium Nitrate.

    Science.gov (United States)

    is reduced by replacing the ammonium nitrate with a solid solution of potassium nitrate in form III ammonium nitrate wherein the potassium nitrate...constitutes from more than zero to less than 50 weight percent of the solid solution . (Author)

  12. Brain propagation of transduced α-synuclein involves non-fibrillar protein species and is enhanced in α-synuclein null mice.

    Science.gov (United States)

    Helwig, Michael; Klinkenberg, Michael; Rusconi, Raffaella; Musgrove, Ruth E; Majbour, Nour K; El-Agnaf, Omar M A; Ulusoy, Ayse; Di Monte, Donato A

    2016-03-01

    Aggregation and neuron-to-neuron transmission are attributes of α-synuclein relevant to its pathogenetic role in human synucleinopathies such as Parkinson's disease. Intraparenchymal injections of fibrillar α-synuclein trigger widespread propagation of amyloidogenic protein species via mechanisms that require expression of endogenous α-synuclein and, possibly, its structural corruption by misfolded conformers acting as pathological seeds. Here we describe another paradigm of long-distance brain diffusion of α-synuclein that involves inter-neuronal transfer of monomeric and/or oligomeric species and is independent of recruitment of the endogenous protein. Targeted expression of human α-synuclein was induced in the mouse medulla oblongata through an injection of viral vectors into the vagus nerve. Enhanced levels of intra-neuronal α-synuclein were sufficient to initiate its caudo-rostral diffusion that likely involved at least one synaptic transfer and progressively reached specific brain regions such as the locus coeruleus, dorsal raphae and amygdala in the pons, midbrain and forebrain. Transfer of human α-synuclein was compared in two separate lines of α-synuclein-deficient mice versus their respective wild-type controls and, interestingly, lack of endogenous α-synuclein expression did not counteract diffusion but actually resulted in a more pronounced and advanced propagation of exogenous α-synuclein. Self-interaction of adjacent molecules of human α-synuclein was detected in both wild-type and mutant mice. In the former, interaction of human α-synuclein with mouse α-synuclein was also observed and might have contributed to differences in protein transmission. In wild-type and α-synuclein-deficient mice, accumulation of human α-synuclein within recipient axons in the pons, midbrain and forebrain caused morphological evidence of neuritic pathology. Tissue sections from the medulla oblongata and pons were stained with different antibodies recognizing

  13. Reducing synuclein accumulation improves neuronal survival after spinal cord injury

    Science.gov (United States)

    Fogerson, Stephanie M.; van Brummen, Alexandra J.; Busch, David J.; Allen, Scott R.; Roychaudhuri, Robin; Banks, Susan M. L.; Klärner, Frank-Gerrit; Schrader, Thomas; Bitan, Gal; Morgan, Jennifer R.

    2016-01-01

    Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson’s and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the “molecular tweezer” inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury. PMID:26854933

  14. Biophysics of α-synuclein membrane interactions.

    Science.gov (United States)

    Pfefferkorn, Candace M; Jiang, Zhiping; Lee, Jennifer C

    2012-02-01

    Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.

  15. α-Synuclein Fibrils Exhibit Gain of Toxic Function, Promoting Tau Aggregation and Inhibiting Microtubule Assembly*

    Science.gov (United States)

    Oikawa, Takayuki; Nonaka, Takashi; Terada, Makoto; Tamaoka, Akira; Hisanaga, Shin-ichi; Hasegawa, Masato

    2016-01-01

    α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells. PMID:27226637

  16. Dopamine Transporter Activity Is Modulated by α-Synuclein.

    Science.gov (United States)

    Butler, Brittany; Saha, Kaustuv; Rana, Tanu; Becker, Jonas P; Sambo, Danielle; Davari, Paran; Goodwin, J Shawn; Khoshbouei, Habibeh

    2015-12-04

    The duration and strength of the dopaminergic signal are regulated by the dopamine transporter (DAT). Drug addiction and neurodegenerative and neuropsychiatric diseases have all been associated with altered DAT activity. The membrane localization and the activity of DAT are regulated by a number of intracellular proteins. α-Synuclein, a protein partner of DAT, is implicated in neurodegenerative disease and drug addiction. Little is known about the regulatory mechanisms of the interaction between DAT and α-synuclein, the cellular location of this interaction, and the functional consequences of this interaction on the basal, amphetamine-induced DAT-mediated dopamine efflux, and membrane microdomain distribution of the transporter. Here, we found that the majority of DAT·α-synuclein protein complexes are found at the plasma membrane of dopaminergic neurons or mammalian cells and that the amphetamine-mediated increase in DAT activity enhances the association of these proteins at the plasma membrane. Further examination of the interaction of DAT and α-synuclein revealed a transient interaction between these two proteins at the plasma membrane. Additionally, we found DAT-induced membrane depolarization enhances plasma membrane localization of α-synuclein, which in turn increases dopamine efflux and enhances DAT localization in cholesterol-rich membrane microdomains.

  17. Dynamic Similarities in Pathological Forms of α-Synuclein

    Science.gov (United States)

    Bradley, Ryan; Maranas, Janna

    2010-03-01

    The natively unstructured, membrane-bound protein α-synuclein is thought to play a role in vesicle trafficking. Its native function is subverted in the pathogenesis of Parkinson's disease, during which it forms fibrillar cytoplasmic aggregates in specific regions in the brain. It is believed that oligomers of α-synuclein are the toxic species, whereas sequestration into fibrils is neuroprotective. Evidence that α-synuclein changes shape as it interacts with membranes suggests that altered dynamics may drive the initial aggregation steps. To test this hypothesis, we conducted separate molecular dynamics simulations of native, mutated, and chemically-damaged forms of α-synuclein, representing the distinct genetic and sporadic causes of the disease. We measured the fractal dimension of individual amino-acid trajectories in order to identify differences in mobility between each simulated protein. Trajectories with higher fractal dimensions are space-filling, and thus correspond to more random, constrained motion; conversely, lower fractal dimensions indicate more directed motions. Although the disease-causing variants of α-synuclein are distinct, they show highly similar dynamical differences from the native form. This suggests that altered dynamics may facilitate oligomerization.

  18. Recent advances in research on Parkinson disease: synuclein and parkin.

    Science.gov (United States)

    Burke, Robert E

    2004-03-01

    Until recently, most research effort on Parkinson disease (PD) was focused on possible environmental causes. With the discovery of mutations in two genes, synuclein and parkin, which are responsible for rare familial forms of the disease, there has been a major change in emphasis. The first genetic cause of PD to be identified was in the gene for synuclein, resulting in an alanine to threonine substitution at position 53. The likely pathogenetic significance of this mutation was supported by the discovery of a second mutation, and the presence of synuclein in Lewy bodies in sporadic PD cases. The synuclein protein has a tendency to self aggregate, and this tendency is increased in the mutants, and by oxidative injury to the protein. While there is growing evidence in animal models that overexpression of wildtype or mutant synuclein may lead to intracytoplasmic inclusions, and dysfunction of dopamine neurons, no animal models in rodents have yet replicated all important features of the disease. Deletions or point mutations in the gene for parkin cause an autosomal recessive, early onset form of parkinsonism. The parkin protein functions as an E3 ubiquitin-protein ligase, and it is involved in the degradation of cellular proteins by the proteasomal pathway. It is hypothesized that the loss of this function results in the toxic accumulation of its target proteins. Research on these inherited forms of PD is pointing towards a common theme, that disturbances of cellular protein handling can lead to the death of dopamine neurons in PD.

  19. Neuropathology of α-synuclein propagation and braak hypothesis.

    Science.gov (United States)

    McCann, Heather; Cartwright, Heidi; Halliday, Glenda M

    2016-02-01

    Parkinson's disease is a progressive neurodegenerative disorder with multiple factors contributing to increasing severity of pathology in specific brain regions. The Braak hypothesis of Lewy pathology progression in Parkinson's disease proposes a systematic spread of α-synuclein that can be staged, with the later stages correlating with clinical aspects of the disease. The spread of pathology through the different stages suggests progression, a theory that has proven correct from evidence of pathology in healthy neurons grafted into the brains of patients with Parkinson's disease. Progression of pathology occurs on a number of levels, within a cell, between nearby cells, and then over longer distances throughout the brain, and evidence using prion proteins suggests two dissociable mechanisms-intracellular toxicity versus a nontoxic infectious mechanism for propagation. In Parkinson's disease, intracellular changes associated with mitochondria and lysosome dysfunction appear important for α-synuclein propagation, with high stress conditions favoring mitochondrial cell death mechanisms. Functional neurons appear necessary for propagation. Unconventional exocytosis releases α-synuclein under stress conditions, and endocytic uptake occurs in nearby cells. This cell-to-cell transmission of α-synuclein has been recapitulated in both cell culture and animal models, but the timeframe of transmission is considerably shorter than that observed in transplanted neurons. The time course of Lewy pathology formation in patients is consistent with the long time course observed in grafted neurons, and the restricted neuronal loss in Parkinson's disease is potentially important for the propagation of α-synuclein through relatively intact circuits.

  20. Interaction of Synuclein and Inflammation in Dopaminergic Neurodegeneration

    Science.gov (United States)

    2014-06-01

    Using the aforementioned cells and several different cell culture experiments with wild-type (WT) and mutated synuclein, we found that both types of...Iwai et al, 1995). To date, three missense mutations in -synuclein (A53T, A30P and E46K) have been noted in PD and these mutations as well as the...COX-2, GFAP, iNOS, NFkB , etc) were up-regulated during the time-course study, the strongest response being between 2 days and four days after the

  1. α-Synuclein oligomers and clinical implications for Parkinson disease.

    Science.gov (United States)

    Kalia, Lorraine V; Kalia, Suneil K; McLean, Pamela J; Lozano, Andres M; Lang, Anthony E

    2013-02-01

    Protein aggregation within the central nervous system has been recognized as a defining feature of neurodegenerative diseases since the early 20th century. Since that time, there has been a growing list of neurodegenerative disorders, including Parkinson disease, which are characterized by inclusions of specific pathogenic proteins. This has led to the long-held dogma that these characteristic protein inclusions, which are composed of large insoluble fibrillar protein aggregates and visible by light microscopy, are responsible for cell death in these diseases. However, the correlation between protein inclusion formation and cytotoxicity is inconsistent, suggesting that another form of the pathogenic proteins may be contributing to neurodegeneration. There is emerging evidence implicating soluble oligomers, smaller protein aggregates not detectable by conventional microscopy, as potential culprits in the pathogenesis of neurodegenerative diseases. The protein α-synuclein is well recognized to contribute to the pathogenesis of Parkinson disease and is the major component of Lewy bodies and Lewy neurites. However, α-synuclein also forms oligomeric species, with certain conformations being toxic to cells. The mechanisms by which these α-synuclein oligomers cause cell death are being actively investigated, as they may provide new strategies for diagnosis and treatment of Parkinson disease and related disorders. Here we review the possible role of α-synuclein oligomers in cell death in Parkinson disease and discuss the potential clinical implications.

  2. Inhibiting α-synuclein oligomerization by stable cell-penetrating β-synuclein fragments recovers phenotype of Parkinson's disease model flies.

    Directory of Open Access Journals (Sweden)

    Ronit Shaltiel-Karyo

    Full Text Available The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we identified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.

  3. Modeled Wet Nitrate Deposition

    Data.gov (United States)

    U.S. Environmental Protection Agency — Modeled data on nitrate wet deposition was obtained from Dr. Jeff Grimm at Penn State Univ. Nitrate wet depostion causes acidification and eutrophication of surface...

  4. Micromachined Amperometric Nitrate Sensor

    OpenAIRE

    Dohyun Kim; Ira Goldberg; Jack Judy

    2003-01-01

    A nitrate-sensing system that consists of a micromachined sensor substrate, nitrate-permeable membrane, integrated microfluidic channels, and standard fluidic connectors has been designed, fabricated, assembled, and tested. Our microsensor was designed for in-situ monitoring of nitrate concentrations in ground water. A silver electrode was patterned for amperometric nitrate detection. An electrochemically oxidized silver electrode was used as a reference electrode. Microfluidic channels were ...

  5. TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity

    DEFF Research Database (Denmark)

    Decressac, Mickael; Mattsson, Bengt; Weikop, Pia

    2013-01-01

    The aggregation of α-synuclein plays a major role in Parkinson disease (PD) pathogenesis. Recent evidence suggests that defects in the autophagy-mediated clearance of α-synuclein contribute to the progressive loss of nigral dopamine neurons. Using an in vivo model of α-synuclein toxicity, we show...... that the PD-like neurodegenerative changes induced by excess cellular levels of α-synuclein in nigral dopamine neurons are closely linked to a progressive decline in markers of lysosome function, accompanied by cytoplasmic retention of transcription factor EB (TFEB), a major transcriptional regulator...... in both A9 and A10 dopamine neurons. Delayed activation of TFEB function through inhibition of mammalian target of rapamycin blocked α-synuclein induced neurodegeneration and further disease progression. The results provide a mechanistic link between α-synuclein toxicity and impaired TFEB function...

  6. Threonine 53 in α-synuclein is conserved in long-living non-primate animals

    DEFF Research Database (Denmark)

    Larsen, Knud; Hedegaard, Claus; Bertelsen, Mads Frost

    2009-01-01

    α-Synuclein is the main constituent of Lewy bodies in familial and sporadic cases of Parkinson's disease (PD). Autosomal dominant point mutations, gene duplications or triplications in the α-synuclein (SNCA) gene cause hereditary forms of PD. One of the α-synuclein point mutations, Ala53Thr......, is associated with increased oligomerization toxicity leading to familial early-onset PD in humans. The amino acid in position 53 in α-synuclein is an alanine in humans, great apes and Old World primates. However, this amino acid is a threonine in the α-synuclein of all other examined species, including New...... World monkeys. Here, we present DNA sequence analysis of SNCA and the deduced amino acid sequences of α-synuclein cloned from various different species, ranging from fish to mammals, which are known for their long-living potential. In all these investigated species the 53Thr is found. We conclude...

  7. 21 CFR 181.33 - Sodium nitrate and potassium nitrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium nitrate and potassium nitrate. 181.33...-Sanctioned Food Ingredients § 181.33 Sodium nitrate and potassium nitrate. Sodium nitrate and potassium nitrate are subject to prior sanctions issued by the U.S. Department of Agriculture for use as sources...

  8. Triptolide Promotes the Clearance of α-Synuclein by Enhancing Autophagy in Neuronal Cells.

    Science.gov (United States)

    Hu, Guanzheng; Gong, Xiaoli; Wang, Le; Liu, Mengru; Liu, Yang; Fu, Xia; Wang, Wei; Zhang, Ting; Wang, Xiaomin

    2016-03-09

    Parkinson's disease (PD) is an aging-associated neurodegenerative disease with a characteristic feature of α-synuclein accumulation. Point mutations (A53T, A30P) that increase the aggregation propensity of α-synuclein result in familial early onset PD. The abnormal metabolism of α-synuclein results in aberrant level changes of α-synuclein in PD. In pathological conditions, α-synuclein is degraded mainly by the autophagy-lysosome pathway. Triptolide (T10) is a monomeric compound isolated from a traditional Chinese herb. Our group demonstrated for the first time that T10 possesses potent neuroprotective properties both in vitro and in vivo PD models. In the present study, we reported T10 as a potent autophagy inducer in neuronal cells, which helped to promote the clearance of various forms of α-synuclein in neuronal cells. We transfected neuronal cells with A53T mutant (A53T) or wild-type (WT) α-synuclein plasmids and found T10 attenuated the cytotoxicity induced by pathogenic A53T α-synuclein overexpression. We observed that T10 significantly reduced both A53T and WT α-synuclein level in neuronal cell line, as well as in primary cultured cortical neurons. Excluding the changes of syntheses, secretion, and aggregation of α-synuclein, we further added autophagy inhibitor or proteasome inhibitor with T10, and we noticed that T10 promoted the clearance of α-synuclein mainly by the autophagic pathway. Lastly, we observed increased autophagy marker LC3-II expression and autophagosomes by GFP-LC3-II accumulation and ultrastructural characterization. However, the lysosome activity and cell viability were not modulated by T10. Our study revealed that T10 could induce autophagy and promote the clearance of both WT and A53T α-synuclein in neurons. These results provide evidence of T10 as a promising mean to treat PD and other neurodegenerative diseases by reducing pathogenic proteins in neurons.

  9. α-Synuclein Fibrils Exhibit Gain of Toxic Function, Promoting Tau Aggregation and Inhibiting Microtubule Assembly.

    Science.gov (United States)

    Oikawa, Takayuki; Nonaka, Takashi; Terada, Makoto; Tamaoka, Akira; Hisanaga, Shin-Ichi; Hasegawa, Masato

    2016-07-15

    α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. α-Synuclein inclusions in the skin of Parkinson's disease and parkinsonism

    OpenAIRE

    2014-01-01

    Objective The presence in the brain of α-synuclein containing Lewy neurites, or bodies, is the histological hallmark of Parkinson's disease (PD). The discovery of α-synuclein aggregates in nerve endings of the heart, digestive tract, and skin has lent support to the concept of PD as a systemic disease. Our goals were, first, to demonstrate the presence of α-synuclein inclusions in the skin and, second, to detect quantitative differences between patients with PD and atypical parkinsonism (AP)....

  11. Induction of α-synuclein aggregate formation by CSF exosomes from patients with Parkinson's disease and dementia with Lewy bodies.

    Science.gov (United States)

    Stuendl, Anne; Kunadt, Marcel; Kruse, Niels; Bartels, Claudia; Moebius, Wiebke; Danzer, Karin M; Mollenhauer, Brit; Schneider, Anja

    2016-02-01

    Extracellular α-synuclein has been proposed as a crucial mechanism for induction of pathological aggregate formation in previously healthy cells. In vitro, extracellular α-synuclein is partially associated with exosomal vesicles. Recently, we have provided evidence that exosomal α-synuclein is present in the central nervous system in vivo. We hypothesized that exosomal α-synuclein species from patients with α-synuclein related neurodegeneration serve as carriers for interneuronal disease transmission. We isolated exosomes from cerebrospinal fluid from patients with Parkinson's disease, dementia with Lewy bodies, progressive supranuclear palsy as a non-α-synuclein related disorder that clinically overlaps with Parkinson's disease, and neurological controls. Cerebrospinal fluid exosome numbers, α-synuclein protein content of cerebrospinal fluid exosomes and their potential to induce oligomerization of α-synuclein were analysed. The quantification of cerebrospinal fluid exosomal α-synuclein showed distinct differences between patients with Parkinson's disease and dementia with Lewy bodies. In addition, exosomal α-synuclein levels correlated with the severity of cognitive impairment in cross-sectional samples from patients with dementia with Lewy bodies. Importantly, cerebrospinal fluid exosomes derived from Parkinson's disease and dementia with Lewy bodies induce oligomerization of α-synuclein in a reporter cell line in a dose-dependent manner. Our data suggest that cerebrospinal fluid exosomes from patients with Parkinson's disease and dementia with Lewy bodies contain a pathogenic species of α-synuclein, which could initiate oligomerization of soluble α-synuclein in target cells and confer disease pathology.

  12. 甲基苯丙胺对SH-SY5Y细胞的毒性作用及对α-突触蛋白表达的影响%THE TOXIC EFFECTS AND THE EXPRESSION OF ALPHA-SYNUCLEIN IN THE SH-SY5Y CELLS TREATED WITH METHAMPHETAMINE

    Institute of Scientific and Technical Information of China (English)

    陈玲; 黄恩平; 台运春; 王慧君; 邱平明

    2013-01-01

    Objective:To investigate METH-induced toxic effects and the expression of α-synuclein in SH-SY5Y cells treated with METH.Methods:SH-SY5Y cells were treated by METH with the concentration of 0,0.5,1.5,2.5,3.5 or 4.5 mmol · L-1 for 24 h.The morphological changes and cell ultrastructure were observed by optical and electron microscopy,respectively.Cell viability and apoptosis rates were assessed by CCK-8 assay and flow cytometry,respectively.The expression levels of α-SN mRNA and protein were detected by quantitative real-time PCR and Western Blot respectively.Results:The SH-SY5Y cells exposed to METH were morphologically featured by shrinkage and dendrite disruption of cells.Autophagosome and inclusion body could be observed in cytoplasm.METH administration caused a dose-dependent reduction in the cell viability (P < 0.001),resulting in an increased cell early apoptotic rate (P < 0.001).The both of α-SN mRNA and protein expression increased with dose-dependent manner in response to METH treatment.Conclusion:Both the cytotoxicity and expression of α-SN in the SH-SY5Y cells are gradually enhanced with the increase of METH concentration.%目的:探讨甲基苯丙胺(methamphetamine,METH)对SH-SY5Y细胞的毒性作用和对α-核突触蛋白(α-synuclein,α-SN)表达的影响,为研究α-SN在METH神经毒性机制中的作用奠定基础.方法:分别用浓度0、0.5、1.5、2.5、3.5、4.5 mmol·L-1的METH处理SH-SYSY细胞24h,倒置显微镜下观察细胞形态变化;透射电镜观察细胞超微结构;CCK-8法检测细胞存活率;流式细胞术分析细胞凋亡率;实时荧光定量PCR和Western Blot分别检测α-SN基因和蛋白水平的表达变化.结果:以0 mmol·L-1为对照组,0.5-4.5 mmol·L-1METH处理的SH-SY5Y细胞镜下可见胞体皱缩变圆,突起变短、断裂、消失.电镜显示,METH处理的细胞内可见包涵体和自噬小体.0.5 mmol·L-1处理组与对照组比较,细胞存活率无显著性差异(P=0.274),其他浓度

  13. Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins

    DEFF Research Database (Denmark)

    Varkey, Jobin; Isas, Jose Mario; Mizuno, Naoko

    2010-01-01

    Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have...... of amphipathic helices alone. Moreover, we frequently observed that a-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that a-synuclein plays a role in vesicle trafficking...

  14. Ammonium nitrate explosion hazards

    Directory of Open Access Journals (Sweden)

    Negovanović Milanka

    2015-01-01

    Full Text Available Ammonium nitrate (AN primarily is used as a fertilizer but it is also very important compound in the production of industrial explosives. The application of ammonium nitrate in the production of industrial explosives was related with the early era of Nobel dynamite and widely increased with the appearance of blasting agents such as ANFO and Slurry, in the middle of the last Century. Throughout the world millions of tons of ammonium nitrate are produced annually and handled without incident. Although ammonium nitrate generally is used safely, accidental explosions involving AN have high impact resulting in loss of lives and destruction of property. The paper presents the basic properties of ammonium nitrate as well as hazards in handling of ammonium nitrate in order to prevent accidents. Several accidents with explosions of ammonium nitrate resulted in catastrophic consequences are listed in the paper as examples of non-compliance with prescribed procedures.

  15. Short-term alpha- or gamma-delta-enriched tocopherol oil supplementation differentially affects the expression of proinflammatory mediators: selective impacts on characteristics of protein tyrosine nitration in vivo

    Directory of Open Access Journals (Sweden)

    Ted H. Elsasser

    2013-10-01

    Full Text Available While vitamin E has been used for decades in cattle diets, the principle form used traditionally is the synthetic α-isoform acetate or succinate and largely no data exist on the biological partitioning or functionality of the major naturally occurring γ- and δ-isoforms in cattle. Using tyrosine 3’-nitrated protein (pNT as a biomarker of nitrosative cell stress, we sought to evaluate the effectiveness of short-term feeding supplementation of high content natural α-tocopherol (α-T, 96% α-isomer compared to high content γ- and δ-enriched low α-content mixed tocopherol oils (γ-T, ~70% γ-, 20% δ-, <5% α-isoform to mitigate systemic and hepatic aspects of the proinflammatory response to endotoxin (LPS. Calves fed diets supplemented with α-T, γ-T for five days or no tocopherol supplement (T0E were challenged with a low-level of LPS (0.25 μg/kg, iv, E. coli 055:B5 sufficient to effect a liver nitration response. As fed, α-T or γ-T increased plasma and liver content of the respective tocopherols reflecting their relative abundance in the respective diets. Plasma or tissue mediators and biomarkers of the proinflammatory response [plasma concentrations of tumor necrosis factor-α (TNF-α, P<0.001, nitrate+nitrite (NOx, P<0.01, and serum amyloid A (SAA, P<0.001], and general liver content of pNT (P<0.005 increased after LPS. LPS-mediated increases in TNF-α were not dif- ferent between diet treatments; both plasma NOx (P<0.05 and generalized liver pNT (P<0.03 responses were attenuated significantly in α-T and γ-T versus T0E calves. Plasma SAA was significantly decreased in γ-T calves at 24 h post-LPS relative to responses in α-T or T0E calves. The nitration of the mitochondrial proteins 24 h post-LPS was not only attenuated in α-T and γ-T vs T0E, but also the mitigating effect of γ-T on these specific nitration events was greater than that of α-T (P<0.01. Results are consistent with the concept that short-term α-T or

  16. α-Synuclein and anti-α-synuclein antibodies in Parkinson's disease, atypical Parkinson syndromes, REM sleep behavior disorder, and healthy controls.

    Directory of Open Access Journals (Sweden)

    Lynnae M Smith

    Full Text Available α-synuclein is thought to play a key role in Parkinson's disease (PD because it is the major protein in Lewy bodies, and because its gene mutations, duplication, and triplication are associated with early-onset PD. There are conflicting reports as to whether serum and plasma concentrations of α-synuclein and anti-α-synuclein antibodies differ between PD and control subjects. The objectives of this study were to compare the levels of α-synuclein and its antibodies between individuals with typical PD (n=14, atypical Parkinson syndromes (n=11, idiopathic rapid eye movement sleep behavior disorder (n=10, and healthy controls (n=9, to assess the strength of association between these serum proteins, and to determine group sizes needed for a high probability (80% power of detecting statistical significance for 25% or 50% differences between typical PD and control subjects for these measurements. Analysis of log-transformed data found no statistically significant differences between groups for either α-synuclein or its antibodies. The concentrations of these proteins were weakly correlated (Spearman rho=0.16. In subjects with typical PD and atypical Parkinson syndromes, anti-α-synuclein antibody levels above 1.5 µg/ml were detected only in subjects with no more than four years of clinical disease. Power analysis indicated that 236 and 73 samples per group would be required for an 80% probability that 25% and 50% differences, respectively, in mean α-synuclein levels between typical PD and control subjects would be statistically significant; for anti-α-synuclein antibodies, 283 and 87 samples per group would be required. Our findings are consistent with those previous studies which suggested that serum concentrations of α-synuclein and its antibodies are not significantly altered in PD.

  17. Defining the oligomerization state of γ-synuclein in solution and in cells.

    Science.gov (United States)

    Golebiewska, Urszula; Zurawsky, Cassandra; Scarlata, Suzanne

    2014-01-21

    γ-Synuclein is expressed at high levels in neuronal cells and in multiple invasive cancers. Like its family member α-synuclein, γ-synuclein is thought to be natively unfolded but does not readily form fibrils. The function of γ-synuclein is unknown, but we have found that it interacts strongly with the enzyme phospholipase Cβ (PLCβ), altering its interaction with G proteins. As a first step in determining its role, we have characterized its oligomerization using fluorescence homotransfer, photon-counting histogram analysis, and native gel electrophoresis. We found that when its expressed in Escherichia coli and purified, γ-synuclein appears monomeric on chromatographs under denaturing conditions, but under native conditions, it appears as oligomers of varying sizes. We followed the monomer-to-tetramer association by labeling the protein with fluorescein and following the concentration-dependent loss in fluorescence anisotropy resulting from fluorescence homotransfer. We also performed photon-counting histogram analysis at increasing concentrations of fluorescein-labeled γ-synuclein and found concentration-dependent oligomerization. Addition of PLCβ2, a strong γ-synuclein binding partner whose cellular expression is correlated with γ-synuclein, results in disruption of γ-synuclein oligomers. Similarly, its binding to lipid membranes promotes the monomer form. When we exogenously express γ-synuclein or microinject purified protein into cells, the protein appears monomeric. Our studies show that even though purified γ-synuclein form oligomers, when binding partners are present, as in cells, it dissociates to a monomer to bind these partners, which in turn may modify protein function and integrity.

  18. Cloud amount/frequency, NITRATE and other data from ALPHA HELIX in the Gulf of Alaska from 1988-09-14 to 1988-09-29 (NCEI Accession 8800279)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The University of Alaska, Institute of Marine Science is responsible for this data collected aboard the R/V Alpha Helix on cruise number HX118 between September 14,...

  19. α-Synuclein vaccination modulates regulatory T cell activation and microglia in the absence of brain pathology

    DEFF Research Database (Denmark)

    Christiansen, Josefine R; Olesen, Mads N; Otzen, Daniel E;

    2016-01-01

    BACKGROUND: Passive and active immunization with α-synuclein has been shown to be neuroprotective in animal models of Parkinson's disease. We have previously shown that vaccination with α-synuclein, long before α-synuclein-induced brain pathology, prevents striatal degeneration by inducing regula...

  20. α-Synuclein aggregation, seeding and inhibition by scyllo-inositol

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, Tarek [Biological Sciences, Sunnybrook Research Institute (Canada); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, M4N 3M5, ON (Canada); McLaurin, JoAnne, E-mail: jmclaurin@sri.utoronto.ca [Biological Sciences, Sunnybrook Research Institute (Canada); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, M4N 3M5, ON (Canada)

    2016-01-15

    Recent literature demonstrates the accelerated aggregation of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease (PD), by the presence of preformed fibrillar conformers in vitro. Furthermore, these preformed fibrillar seeds are suggested to accelerate pathological induction in vivo when injected into the brains of mice. Variation in the results of in vivo studies is proposed to be caused by α-synuclein conformational variants. To investigate the impact of amino acid sequence on seeding efficiency, human and mouse α-synuclein seeds, which vary at 7 amino acid residues, were generated and cross-seeding kinetics studied. Using transmission electron microscopy (TEM), we confirmed that mouse α-synuclein aggregated more rapidly than human α-synuclein. Subsequently, we determined that seeding of human and mouse α-synuclein was more rapid in the presence of seeds generated from the same species. In addition, an established amyloid inhibitor, scyllo-inositol, was examined for potential inhibitory effects on α-synuclein aggregation. TEM analysis of protein:inhibitor assays demonstrated that scyllo-inositol inhibits the aggregation of α-synuclein, suggesting the therapeutic potential of the small molecule in PD. - Highlights: • Mouse α-syn fibrillizes in a significantly shorter timeframe than human α-syn. • Seeding of monomers is more efficient when seeds originate from the same species. • scyllo-Inositol has anti-aggregation effects on mouse and human α-syn.

  1. Insulin-degrading enzyme prevents α-synuclein fibril formation in a nonproteolytical manner.

    Science.gov (United States)

    Sharma, Sandeep K; Chorell, Erik; Steneberg, Pär; Vernersson-Lindahl, Emma; Edlund, Helena; Wittung-Stafshede, Pernilla

    2015-07-31

    The insulin-degrading enzyme (IDE) degrades amyloidogenic proteins such as Amyloid β (Αβ) and Islet Amyloid Polypeptide (IAPP), i.e. peptides associated with Alzheimer's disease and type 2 diabetes, respectively. In addition to the protease activity normally associated with IDE function an additional activity involving the formation of stable, irreversible complexes with both Αβ and α-synuclein, an amyloidogenic protein involved in Parkinson's disease, was recently proposed. Here, we have investigated the functional consequences of IDE-α-synuclein interactions in vitro. We demonstrate that IDE in a nonproteolytic manner and at sub-stoichiometric ratios efficiently inhibits α-synuclein fibril formation by binding to α-synuclein oligomers making them inert to amyloid formation. Moreover, we show that, within a defined range of α-synuclein concentrations, interaction with α-synuclein oligomers increases IDE's proteolytic activity on a fluorogenic substrate. We propose that the outcomes of IDE-α-synuclein interactions, i.e. protection against α-synuclein amyloid formation and stimulated IDE protease activity, may be protective in vivo.

  2. Cholesterol facilitates interactions between α-synuclein oligomers and charge-neutral membranes

    DEFF Research Database (Denmark)

    van Maarschalkerweerd, Andreas; Vetri, Valeria; Vestergaard, Bente

    2015-01-01

    composed of anionic lipids, while the more physiologically relevant zwitterionic lipids remain intact. We present experimental evidence for significant morphological changes in zwitterionic membranes containing cholesterol, induced by α-synuclein oligomers. Depending on the lipid composition, model...... of cholesterol for mediating interactions between physiologically relevant membranes and α-synuclein....

  3. α-Synuclein aggregation, seeding and inhibition by scyllo-inositol.

    Science.gov (United States)

    Ibrahim, Tarek; McLaurin, JoAnne

    2016-01-15

    Recent literature demonstrates the accelerated aggregation of α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease (PD), by the presence of preformed fibrillar conformers in vitro. Furthermore, these preformed fibrillar seeds are suggested to accelerate pathological induction in vivo when injected into the brains of mice. Variation in the results of in vivo studies is proposed to be caused by α-synuclein conformational variants. To investigate the impact of amino acid sequence on seeding efficiency, human and mouse α-synuclein seeds, which vary at 7 amino acid residues, were generated and cross-seeding kinetics studied. Using transmission electron microscopy (TEM), we confirmed that mouse α-synuclein aggregated more rapidly than human α-synuclein. Subsequently, we determined that seeding of human and mouse α-synuclein was more rapid in the presence of seeds generated from the same species. In addition, an established amyloid inhibitor, scyllo-inositol, was examined for potential inhibitory effects on α-synuclein aggregation. TEM analysis of protein:inhibitor assays demonstrated that scyllo-inositol inhibits the aggregation of α-synuclein, suggesting the therapeutic potential of the small molecule in PD.

  4. Piceatannol and Other Wine Stilbenes: A Pool of Inhibitors against α-Synuclein Aggregation and Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Hamza Temsamani

    2016-06-01

    Full Text Available The aggregation of α-synuclein is one on the key pathogenic events in Parkinson’s disease. In the present study, we investigated the inhibitory capacities of stilbenes against α-synuclein aggregation and toxicity. Thioflavin T fluorescence, transmission electronic microscopy, and SDS-PAGE analysis were performed to investigate the inhibitory effects of three stilbenes against α-synuclein aggregation: piceatannol, ampelopsin A, and isohopeaphenol. Lipid vesicle permeabilization assays were performed to screen stilbenes for protection against membrane damage induced by aggregated α-synuclein. The viability of PC12 cells was examined using an MTT assay to assess the preventive effects of stilbenes against α-synuclein-induced toxicity. Piceatannol inhibited the formation of α synuclein fibrils and was able to destabilize preformed filaments. It seems to induce the formation of small soluble complexes protecting membranes against α-synuclein-induced damage. Finally, piceatannol protected cells against α-synuclein-induced toxicity. The oligomers tested (ampelopsin A and hopeaphenol were less active.

  5. Sensitive Electrochemical Detection of Native and Aggregated -Synuclein Protein Involved in Parkinson's Disease

    NARCIS (Netherlands)

    Masarik, Michal; Stobiecka, Agata; Kizek, René; Jelen, Frantisek; Pechan, Zdenk; Hoyer, Wolfgang; Subramaniam, Vinod; Palecek, Emil

    2004-01-01

    The aggregation of α-synuclein, a 14 kDa protein, is involved in several human neurodegenerative disorders, including Parkinson's disease. We studied native and in vitro aggregated α-synuclein by circular dichroism (CD), atomic force microscopy (AFM) and electrochemical methods. We used constant cur

  6. Mannose 6-Phosphate Receptor Is Reduced in -Synuclein Overexpressing Models of Parkinsons Disease

    DEFF Research Database (Denmark)

    Matrone, Carmela; Dzamko, Nicolas; Madsen, Peder;

    2016-01-01

    risk of neurodegeneration. Defects in cathepsin D (CD) processing and α-synuclein degradation causing its accumulation in lysosomes are particularly relevant for the development of Parkinson's disease (PD). However, the mechanism by which alterations in CD maturation and α-synuclein degradation leads...

  7. Agricultural nitrate pollution

    DEFF Research Database (Denmark)

    Anker, Helle Tegner

    2015-01-01

    Despite the passing of almost 25 years since the adoption of the EU Nitrates Directive, agricultural nitrate pollution remains a major concern in most EU Member States. This is also the case in Denmark, although a fairly strict regulatory regime has resulted in almost a 50 per cent reduction...

  8. Nitrate Leaching Index

    Science.gov (United States)

    The Nitrate Leaching Index is a rapid assessment tool that evaluates nitrate (NO3) leaching potential based on basic soil and climate information. It is the basis for many nutrient management planning efforts, but it has considerable limitations because of : 1) an oversimplification of the processes...

  9. Alpha Thalassemia

    Science.gov (United States)

    Alpha Thalassemia Physicians often mistake alpha thalassemia trait for iron deficiency anemia and incorrectly prescribe iron supplements that have no effect 1 on the anemia. αα αα Normal alpha ...

  10. Molecular mechanism of abnormal aggregation of α-synuclein

    Institute of Scientific and Technical Information of China (English)

    HU HongYua; LIN XiaoJing

    2007-01-01

    The abnormal aggregation of α-synuclein (α-Syn) is thought to be closely associated with Parkinson's disease, but the pathogenesis is still unclear. In this review, we survey the latest development in the molecular mechanism of abnormal α-Syn aggregation, especially in the aspects of the core sequences, aggregation inhibitors, structural transformation and filament morphologies. By exploring the mechanism of α-Syn aggregation, we will have a better understanding of the disease pathogenesis, and develop strategies for preventing and treating this severe disease.

  11. Seeking a Mechanism for the Toxicity of Oligomeric α-Synuclein

    Directory of Open Access Journals (Sweden)

    Hazel L. Roberts

    2015-03-01

    Full Text Available In a number of neurological diseases including Parkinson’s disease (PD, α‑synuclein is aberrantly folded, forming abnormal oligomers, and amyloid fibrils within nerve cells. Strong evidence exists for the toxicity of increased production and aggregation of α-synuclein in vivo. The toxicity of α-synuclein is popularly attributed to the formation of “toxic oligomers”: a heterogenous and poorly characterized group of conformers that may share common molecular features. This review presents the available evidence on the properties of α-synuclein oligomers and the potential molecular mechanisms of their cellular disruption. Toxic α-synuclein oligomers may impact cells in a number of ways, including the disruption of membranes, mitochondrial depolarization, cytoskeleton changes, impairment of protein clearance pathways, and enhanced oxidative stress. We also examine the relationship between α-synuclein toxic oligomers and amyloid fibrils, in the light of recent studies that paint a more complex picture of α-synuclein toxicity. Finally, methods of studying and manipulating oligomers within cells are described.

  12. New roles of glycosaminoglycans in α-synuclein aggregation in a cellular model of Parkinson disease.

    Directory of Open Access Journals (Sweden)

    Sonia Lehri-Boufala

    Full Text Available The causes of Parkinson disease (PD remain mysterious, although some evidence supports mitochondrial dysfunctions and α-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of α-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD, the major lysosomal protease responsible of α-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in α-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neuroblastoma cell model of PD induced by MPP+, the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of α-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited α-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway, regulating cathD activity and affecting two main biological processes, α-synuclein aggregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology.

  13. New Roles of Glycosaminoglycans in α-Synuclein Aggregation in a Cellular Model of Parkinson Disease

    Science.gov (United States)

    Lehri-Boufala, Sonia; Ouidja, Mohand-Ouidir; Barbier-Chassefière, Véronique; Hénault, Emilie; Raisman-Vozari, Rita; Garrigue-Antar, Laure; Papy-Garcia, Dulce; Morin, Christophe

    2015-01-01

    The causes of Parkinson disease (PD) remain mysterious, although some evidence supports mitochondrial dysfunctions and α-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of α-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD), the major lysosomal protease responsible of α-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs) regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in α-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neuroblastoma cell model of PD induced by MPP+, the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of α-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited α-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway, regulating cathD activity and affecting two main biological processes, α-synuclein aggregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology. PMID:25617759

  14. Inhibition effects of tanshinone on the aggregation of α-synuclein.

    Science.gov (United States)

    Ji, Kaige; Zhao, Yudan; Yu, Tianhong; Wang, Zhuoyi; Gong, Hao; Yang, Xin; Liu, Yang; Huang, Kun

    2016-01-01

    Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Lewy bodies that are formed by the aggregated α-synuclein are a major pathological feature of PD. Salvia miltiorrhiza has been used as food and as a traditional medicine for centuries in China, with tanshinone I (TAN I) and tanshinone IIA (TAN IIA) as its major bioactive ingredients. Here, we investigated the effects of TAN I and TAN IIA on α-synuclein aggregation both in vitro and in a transgenic Caenorhabditis elegans PD model (NL5901). We demonstrated that TAN I and TAN IIA inhibited the aggregation of α-synuclein as demonstrated by the prolonged lag time and the reduced thioflavin-T fluorescence intensity; TAN I and TAN IIA also disaggregated preformed mature fibrils in vitro. Moreover, the presence of TAN I or TAN IIA affected the secondary structural transformation of α-synuclein from unstructured coils to β-sheets, and alleviated the membrane disruption caused by aggregated α-synuclein in vitro. Besides, the immuno-dot-blot assay indicated that TAN I and TAN IIA reduce the formation of oligomers and fibrils. We further found that TAN I and TAN IIA extended the life span of NL5901, a strain of transgenic C. elegans that expresses human α-synuclein, possibly by attenuating the aggregation of α-synuclein. Taken together, our results suggested that TAN I and TAN IIA may be explored further as potential candidates for the prevention and treatment of PD.

  15. Insulin-degrading enzyme is activated by the C-terminus of α-synuclein.

    Science.gov (United States)

    Sharma, Sandeep K; Chorell, Erik; Wittung-Stafshede, Pernilla

    2015-10-16

    The insulin-degrading enzyme (IDE) plays a key role in type-2 diabetes and typically degrades small peptides such as insulin, amyloid β and islet amyloid polypeptide. We recently reported a novel non-proteolytical interaction in vitro between IDE and the Parkinson's disease 140-residue protein α-synuclein that resulted in dual effects: arrested α-synuclein oligomers and, simultaneously, increased IDE proteolysis activity. Here we demonstrate that these outcomes arise due to IDE interactions with the C-terminus of α-synuclein. Whereas a peptide containing the first 97 residues of α-synuclein did not improve IDE activity and its aggregation was not blocked by IDE, a peptide with the C-terminal 44 residues of α-synuclein increased IDE proteolysis to the same degree as full-length α-synuclein. Because the α-synuclein C-terminus is acidic, the interaction appears to involve electrostatic attraction with IDE's basic exosite, known to be involved in activation.

  16. α-Synuclein overexpression increases dopamine toxicity in BE(2-M17 cells

    Directory of Open Access Journals (Sweden)

    Miller David W

    2010-03-01

    Full Text Available Abstract Background Oxidative stress has been proposed to be involved in the pathogenesis of Parkinson's disease (PD. A plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine and produce various toxic molecules, i.e., free radicals and quinone species. α-Synuclein, a protein found in Lewy bodies characteristic of PD, is also thought to be involved in the pathogenesis of PD and point mutations and multiplications in the gene coding for α-synuclein have been found in familial forms of PD. Results We used dopaminergic human neuroblastoma BE(2-M17 cell lines stably transfected with WT or A30P mutant α-synuclein to characterize the effect of α-synuclein on dopamine toxicity. Cellular toxicity was analyzed by lactate dehydrogenase assay and by fluorescence-activated cell sorter analysis. Increased expression of either wild-type or mutant α-synuclein enhances the cellular toxicity induced by the accumulation of intracellular dopamine or DOPA. Conclusions Our results suggest that an interplay between dopamine and α-synuclein can cause cell death in a neuron-like background. The data presented here are compatible with several models of cytotoxicity, including the formation of α-synuclein oligomers and impairment of the lysosomal degradation.

  17. Membrane Permeabilization by Oligomeric α-Synuclein: In Search of the Mechanism.

    Directory of Open Access Journals (Sweden)

    Bart D van Rooijen

    Full Text Available BACKGROUND: The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinson's disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory. METHODOLOGY/PRINCIPAL FINDINGS: Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general. CONCLUSIONS/SIGNIFICANCE: The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by

  18. Bioactivation of organic nitrates and the mechanism of nitrate tolerance.

    Science.gov (United States)

    Klemenska, Emila; Beresewicz, Andrzej

    2009-01-01

    Organic nitrates, such as nitroglycerin, are commonly used in the therapy of cardiovascular disease. Long-term therapy with these drugs, however, results in the rapid development of nitrate tolerance, limiting their hemodynamic and anti-ischemic efficacy. In addition, nitrate tolerance is associated with the expression of potentially deleterious modifications such as increased oxidative stress, endothelial dysfunction, and sympathetic activation. In this review we discuss current concepts regarding the mechanisms of organic nitrate bioactivation, nitrate tolerance, and nitrate-mediated oxidative stress and endothelial dysfunction. We also examine how hydralazine may prevent nitrate tolerance and related endothelial dysfunction.

  19. Formation of covalent di-tyrosine dimers in recombinant α-synuclein

    DEFF Research Database (Denmark)

    van Maarschalkerweerd, A; Pedersen, MN; Peterson, H

    2015-01-01

    Parkinson's disease is associated with fibril deposition in the diseased brain. Misfolding events of the intrinsically disordered synaptic protein α-synuclein are suggested to lead to the formation of transient oligomeric and cytotoxic species. The etiology of Parkinson's disease is further...... associated with mitochondrial dysfunction and formation of reactive oxygen species. Oxidative stress causes chemical modification of native α-synuclein, plausibly further influencing misfolding events. Here, we present evidence for the spontaneous formation of covalent di-tyrosine α-synuclein dimers...

  20. α-Synuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity*♦

    Science.gov (United States)

    Chen, Robert H. C.; Wislet-Gendebien, Sabine; Samuel, Filsy; Visanji, Naomi P.; Zhang, Gang; Marsilio, Diana; Langman, Tammy; Fraser, Paul E.; Tandon, Anurag

    2013-01-01

    α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity. PMID:23344955

  1. VT Nitrate Leaching Index

    Data.gov (United States)

    Vermont Center for Geographic Information — (Link to Metadata) Nitrate Leaching Index data for the state of Vermont. This is a derivative product based on the SSURGO soils data for all counties except Essex...

  2. Agricultural nitrate pollution

    DEFF Research Database (Denmark)

    Anker, Helle Tegner

    2015-01-01

    Despite the passing of almost 25 years since the adoption of the EU Nitrates Directive, agricultural nitrate pollution remains a major concern in most EU Member States. This is also the case in Denmark, although a fairly strict regulatory regime has resulted in almost a 50 per cent reduction...... in nitrogen leaching since the mid-80s. Nevertheless, further effort is needed, particularly in ecologically sensitive areas. This article discusses different regulatory approaches – and in particular the need for a differentiated nitrate regulation tailored to meet site-specific ecological demands – from...... of the mandatory specification standards of the Nitrates Directive combined with additional instruments to address the need for severe restrictions on fertiliser use or cultivation practices in the most ecologically vulnerable areas....

  3. Nitrate storage and dissimilatory nitrate reduction by eukaryotic microbes

    DEFF Research Database (Denmark)

    Kamp, Anja; Høgslund, Signe; Risgaard-Petersen, Nils

    2015-01-01

    and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate....... A first compilation of intracellular nitrate inventories in various marine sediments is presented, indicating that intracellular nitrate pools vastly exceed porewater nitrate pools. The relative contribution by foraminifers to total sedimentary denitrification is estimated for different marine settings...

  4. Protein tyrosine nitration

    Science.gov (United States)

    Chaki, Mounira; Leterrier, Marina; Barroso, Juan B

    2009-01-01

    Nitric oxide metabolism in plant cells has a relative short history. Nitration is a chemical process which consists of introducing a nitro group (-NO2) into a chemical compound. in biological systems, this process has been found in different molecules such as proteins, lipids and nucleic acids that can affect its function. This mini-review offers an overview of this process with special emphasis on protein tyrosine nitration in plants and its involvement in the process of nitrosative stress. PMID:19826215

  5. The Aggregation of Huntingtin and α-Synuclein

    Directory of Open Access Journals (Sweden)

    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.

  6. Tryptophan probes at the α-synuclein and membrane interface

    Science.gov (United States)

    Pfefferkorn, Candace M.; Lee, Jennifer C.

    2010-01-01

    Understanding how environmental factors affect the conformational dynamics of α-synuclein (α-syn) is of great importance because the accumulation and deposit of aggregated α-syn in the brain are intimately connected to Parkinson’s disease etiology. Measurements of steady-state and time-resolved fluorescence of single tryptophan-containing α-syn variants have revealed distinct phospholipid vesicle and micelle interactions at residues 4, 39, 94, and 125. Our circular dichroism (CD) data confirm that Trp mutations do not affect α-syn membrane binding properties (apparent association constant Kaapp∼1×107M−1 for all synucleins) saturating at an estimated lipid-to-protein molar ratio of 380 or approximately 120 proteins covering ~7% of the surface area of an 80 nm diameter vesicle. Fluorophores at positions 4 and 94 are the most sensitive to the lipid bilayer with pronounced spectral blue-shifts (W4: Δλmax ~23 nm; W94: Δλmax ~10 nm) and quantum yield increases (W4, W94: ~3 fold) while W39 and W125 remain primarily water-exposed. Time-resolved fluorescence data show that all sites (except W125) have subpopulations that interact with the membrane. PMID:20229987

  7. Mechanistic study of the inhibitory activity of Geum urbanum extract against α-Synuclein fibrillation

    DEFF Research Database (Denmark)

    Lobbens, Eva Stephanie; Breydo, Leonid; Pedersen, Thomas Skamris;

    2016-01-01

    The presence of Lewy bodies and Lewy neurites is a major pathological hallmark of Parkinson's disease and is hypothesized to be linked to disease development, although this is not yet conclusive. Lewy bodies and Lewy neurites primarily consist of fibrillated α-Synuclein; yet, there is no treatment...... available targeting stabilization of α-Synuclein in its native state. The aim of the present study was to investigate the inhibitory activity of an ethanolic extract of Geum urbanum against α-Synuclein fibrillation and examine the structural changes of α-Synuclein in the presence of the extract. The anti......-fibrillation and anti aggregation activities of the plant extract were monitored by thioflavin T fibrillation assays and size exclusion chromatography, while structural changes were followed by circular dichroism, Fourier transform infrared spectroscopy, intrinsic fluorescence, small angle X-ray scattering and electron...

  8. Structural Investigations of on-pathway Oligomers of α-Synuclein

    DEFF Research Database (Denmark)

    Pedersen, Martin Nors; Horvath, Istvan; Weise, Christoph F.;

    Here we present Small Angle X-ray (SAXS) data of α-synuclein oligomers obtained by incubation with the ligand FN075. Data from complementary methods such as NMR and CD are also shown. Aggregated α-synuclein is the major constituent of the Lewy Bodies regarded as the hallmark of Parkinson’s Disease...... and Dementia with Lewy Bodies (DLB) (Spillantini et al. 1997). The role of lewy bodies in the pathology of Parkinson’s Disease and DLB is however not well understood but in vitro experiments suggest that transient oligomeric species could be involved in cell toxicity (Giehm et al. 2011). The natural function...... of α-synuclein has also not been established (Drescher et al. 2012). The monomeric species of α-synuclein is intrinsically disordered meaning it does not have just one stable conformation in solution. The solution structure of an on pathway oligomer consisting of 16 monomers has been solved...

  9. Mannose 6-Phosphate Receptor Is Reduced in -Synuclein Overexpressing Models of Parkinsons Disease

    DEFF Research Database (Denmark)

    Matrone, Carmela; Dzamko, Nicolas; Madsen, Peder;

    2016-01-01

    Increasing evidence points to defects in autophagy as a common denominator in most neurodegenerative conditions. Progressive functional decline in the autophagy-lysosomal pathway (ALP) occurs with age, and the consequent impairment in protein processing capacity has been associated with a higher...... to autophagy defects in PD neurons is still uncertain. Here we demonstrate that MPR300 shuttling between endosomes and the trans Golgi network is altered in α-synuclein overexpressing neurons. Consequently, CD is not correctly trafficked to lysosomes and cannot be processed to generate its mature active form......, leading to a reduced CD-mediated α-synuclein degradation and α-synuclein accumulation in neurons. MPR300 is downregulated in brain from α-synuclein overexpressing animal models and in PD patients with early diagnosis. These data indicate MPR300 as crucial player in the autophagy-lysosomal dysfunctions...

  10. Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism.

    Science.gov (United States)

    Prusiner, Stanley B; Woerman, Amanda L; Mordes, Daniel A; Watts, Joel C; Rampersaud, Ryan; Berry, David B; Patel, Smita; Oehler, Abby; Lowe, Jennifer K; Kravitz, Stephanie N; Geschwind, Daniel H; Glidden, David V; Halliday, Glenda M; Middleton, Lefkos T; Gentleman, Steve M; Grinberg, Lea T; Giles, Kurt

    2015-09-22

    Prions are proteins that adopt alternative conformations that become self-propagating; the PrP(Sc) prion causes the rare human disorder Creutzfeldt-Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T-YFP) and TgM83(+/-) mice expressing α-synuclein (A53T). The TgM83(+/-) mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83(+/+) mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83(+/-) mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T-YFP in cultured cells, whereas none of six Parkinson's disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83(+/+) mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.

  11. Neurodegenerative phenotypes in an A53T α-synuclein transgenic mouse model are independent of LRRK2

    Science.gov (United States)

    Daher, João Paulo L.; Pletnikova, Olga; Biskup, Saskia; Musso, Alessandra; Gellhaar, Sandra; Galter, Dagmar; Troncoso, Juan C.; Lee, Michael K.; Dawson, Ted M.; Dawson, Valina L.; Moore, Darren J.

    2012-01-01

    Mutations in the genes encoding LRRK2 and α-synuclein cause autosomal dominant forms of familial Parkinson's disease (PD). Fibrillar forms of α-synuclein are a major component of Lewy bodies, the intracytoplasmic proteinaceous inclusions that are a pathological hallmark of idiopathic and certain familial forms of PD. LRRK2 mutations cause late-onset familial PD with a clinical, neurochemical and, for the most part, neuropathological phenotype that is indistinguishable from idiopathic PD. Importantly, α-synuclein-positive Lewy bodies are the most common pathology identified in the brains of PD subjects harboring LRRK2 mutations. These observations may suggest that LRRK2 functions in a common pathway with α-synuclein to regulate its aggregation. To explore the potential pathophysiological interaction between LRRK2 and α-synuclein in vivo, we modulated LRRK2 expression in a well-established human A53T α-synuclein transgenic mouse model with transgene expression driven by the hindbrain-selective prion protein promoter. Deletion of LRRK2 or overexpression of human G2019S-LRRK2 has minimal impact on the lethal neurodegenerative phenotype that develops in A53T α-synuclein transgenic mice, including premature lethality, pre-symptomatic behavioral deficits and human α-synuclein or glial neuropathology. We also find that endogenous or human LRRK2 and A53T α-synuclein do not interact together to influence the number of nigrostriatal dopaminergic neurons. Taken together, our data suggest that α-synuclein-related pathology, which occurs predominantly in the hindbrain of this A53T α-synuclein mouse model, occurs largely independently from LRRK2 expression. These observations fail to provide support for a pathophysiological interaction of LRRK2 and α-synuclein in vivo, at least within neurons of the mouse hindbrain. PMID:22357653

  12. MIDBRAIN CATECHOLAMINERGIC NEURONS CO-EXPRESS α-SYNUCLEIN AND TAU IN PROGRESSIVE SUPRANUCLEAR PALSY

    Directory of Open Access Journals (Sweden)

    María Elena eErro Aguirre

    2015-03-01

    Full Text Available Objective: To analyze the frequency and distribution of α-synuclein deposits in progressive supranuclear palsy (PSP.Methods: The brains of 25 cases of pathologically confirmed PSP were evaluated with immunohistochemistry for α-synuclein and tau. Multiple immunofluorescent stains were applied to analyze the expression of tau and α-synuclein aggregates in catecholaminergic neurons. Patients’ clinical symptoms were retrospectively recorded. Results: Deposits α-synuclein in the form of typical Lewy bodies (LBs were only found in two PSP cases (8% that fulfilled the clinical subtype of PSP known as Richardson’s syndrome (RS. LBs were present in the locus ceruleus, substantia nigra pars compacta, basal forebrain, amygdala and cingulated cortex in a distribution mimicking that of Parkinson’s disease. Triple-immunolabeling revealed co-expression of α-synuclein and tau proteins in some tyrosine hydroxilase-positive neurons of the locus ceruleus and substantia nigra pars compacta.Conclusions: There is no apparent clinical correlation between the presence of LBs in PSP. Tau protein co-aggregate with α-synuclein in catecholaminergic neurons of PSP brains suggesting a synergistic interaction between the two proteins. This is in keeping with the current view of neurodegenerative disorders as ‘misfolded protein diseases’.

  13. Curcumin Treatment Improves Motor Behavior in α-Synuclein Transgenic Mice.

    Directory of Open Access Journals (Sweden)

    Kateri J Spinelli

    Full Text Available The curry spice curcumin plays a protective role in mouse models of neurodegenerative diseases, and can also directly modulate aggregation of α-synuclein protein in vitro, yet no studies have described the interaction of curcumin and α-synuclein in genetic synucleinopathy mouse models. Here we examined the effect of chronic and acute curcumin treatment in the Syn-GFP mouse line, which overexpresses wild-type human α-synuclein protein. We discovered that curcumin diet intervention significantly improved gait impairments and resulted in an increase in phosphorylated forms of α-synuclein at cortical presynaptic terminals. Acute curcumin treatment also caused an increase in phosphorylated α-synuclein in terminals, but had no direct effect on α-synuclein aggregation, as measured by in vivo multiphoton imaging and Proteinase-K digestion. Using LC-MS/MS, we detected ~5 ng/mL and ~12 ng/mL free curcumin in the plasma of chronic or acutely treated mice, with a glucuronidation rate of 94% and 97%, respectively. Despite the low plasma levels and extensive metabolism of curcumin, these results show that dietary curcumin intervention correlates with significant behavioral and molecular changes in a genetic synucleinopathy mouse model that mimics human disease.

  14. An extract of the marine alga Alaria esculenta modulates α-synuclein folding and amyloid formation.

    Science.gov (United States)

    Giffin, James C; Richards, Robert C; Craft, Cheryl; Jahan, Nusrat; Leggiadro, Cindy; Chopin, Thierry; Szemerda, Michael; MacKinnon, Shawna L; Ewart, K Vanya

    2017-02-23

    The conversion of α-synuclein from its natively unfolded and α-helical tetrameric forms to an amyloid conformation is central to the emergence of Parkinson's disease. Therefore, prevention of this conversion may offer an effective way of avoiding the onset of this disease or delaying its progress. At different concentrations, an aqueous extract from the edible winged kelp (Alaria esculenta), was shown to lower and to raise the melting point of α-synuclein. Size fractionation of the extract resulted in the separation of these distinct activities. The fraction below 5kDa decreased the melting point of α-synuclein, whereas the fraction above 10kDa raised the melting point. Both of these fractions were found to inhibit the formation of amyloid aggregates by α-synuclein, measured by thioflavin T dye-binding assays; this effect was further confirmed by transmission electron microscopy showing the inhibition of fibril formation. Circular dichroism analysis suggested that the incubation of α-synuclein under fibrillation conditions resulted in the loss of substantial native helical structure in the presence and absence of the fractions. It is therefore likely that the fractions inhibit fibrillation by interacting with the unfolded form of α-synuclein.

  15. Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

    Institute of Scientific and Technical Information of China (English)

    Feng ZHANG; Li-Na JI; Lin TANG; Jun HU; Hong-Yu HU; Hong-Jie XU; Jian-Hua HE

    2005-01-01

    Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerafive diseases such as Parkinson's disease, dementia with Lewy bodies and Lewy body variant of Alzheimer's disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1 h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how β-sheets assemble into α-synuclein fibrils on a nanometer scale.

  16. Mannose 6-Phosphate Receptor Is Reduced in -Synuclein Overexpressing Models of Parkinsons Disease

    Science.gov (United States)

    Matrone, Carmela; Dzamko, Nicolas; Madsen, Peder; Nyegaard, Mette; Pohlmann, Regina; Søndergaard, Rikke V.; Lassen, Louise B.; Andresen, Thomas L.; Halliday, Glenda M.; Jensen, Poul Henning

    2016-01-01

    Increasing evidence points to defects in autophagy as a common denominator in most neurodegenerative conditions. Progressive functional decline in the autophagy-lysosomal pathway (ALP) occurs with age, and the consequent impairment in protein processing capacity has been associated with a higher risk of neurodegeneration. Defects in cathepsin D (CD) processing and α-synuclein degradation causing its accumulation in lysosomes are particularly relevant for the development of Parkinson's disease (PD). However, the mechanism by which alterations in CD maturation and α-synuclein degradation leads to autophagy defects in PD neurons is still uncertain. Here we demonstrate that MPR300 shuttling between endosomes and the trans Golgi network is altered in α-synuclein overexpressing neurons. Consequently, CD is not correctly trafficked to lysosomes and cannot be processed to generate its mature active form, leading to a reduced CD-mediated α-synuclein degradation and α-synuclein accumulation in neurons. MPR300 is downregulated in brain from α-synuclein overexpressing animal models and in PD patients with early diagnosis. These data indicate MPR300 as crucial player in the autophagy-lysosomal dysfunctions reported in PD and pinpoint MRP300 as a potential biomarker for PD. PMID:27509067

  17. Altered α-synuclein, parkin, and synphilin isoform levels in multiple system atrophy brains

    DEFF Research Database (Denmark)

    Brudek, Tomasz; Winge, Kristian; Rasmussen, Nadja Bredo

    2016-01-01

    Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease-specific transcript......Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease......-specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α-synucleinopathies. In this study, the differential expression of α-synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD...... for parkin and synphilin-1 isoforms. In MSA brains, α-synuclein140 and α-synuclein 112 isoform levels were significantly increased, whereas levels of the α-synuclein 126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus versus controls. Moreover, in MSA cases...

  18. α-Synuclein increases β-amyloid secretion by promoting β-/γ-secretase processing of APP.

    Science.gov (United States)

    Roberts, Hazel L; Schneider, Bernard L; Brown, David R

    2017-01-01

    α-Synuclein misfolding and aggregation is often accompanied by β-amyloid deposition in some neurodegenerative diseases. We hypothesised that α-synuclein promotes β-amyloid production from APP. β-Amyloid levels and APP amyloidogenic processing were investigated in neuronal cell lines stably overexpressing wildtype and mutant α-synuclein. γ-Secretase activity and β-secretase expression were also measured. We show that α-synuclein expression induces β-amyloid secretion and amyloidogenic processing of APP in neuronal cell lines. Certain mutations of α-synuclein potentiate APP amyloidogenic processing. γ-Secretase activity was not enhanced by wildtype α-synuclein expression, however β-secretase protein levels were induced. Furthermore, a correlation between α-synuclein and β-secretase protein was seen in rat brain striata. Iron chelation abolishes the effect of α-synuclein on neuronal cell β-amyloid secretion, whereas overexpression of the ferrireductase enzyme Steap3 is robustly pro-amyloidogenic. We propose that α-synuclein promotes β-amyloid formation by modulating β-cleavage of APP, and that this is potentially mediated by the levels of reduced iron and oxidative stress.

  19. Increased lipolysis and altered lipid homeostasis protect γ-synuclein-null mutant mice from diet-induced obesity.

    Science.gov (United States)

    Millership, Steven; Ninkina, Natalia; Guschina, Irina A; Norton, Jessica; Brambilla, Riccardo; Brambilla, Ricardo; Oort, Pieter J; Adams, Sean H; Dennis, Rowena J; Voshol, Peter J; Rochford, Justin J; Buchman, Vladimir L

    2012-12-18

    Synucleins are a family of homologous proteins principally known for their involvement in neurodegeneration. γ-Synuclein is highly expressed in human white adipose tissue and increased in obesity. Here we show that γ-synuclein is nutritionally regulated in white adipose tissue whereas its loss partially protects mice from high-fat diet (HFD)-induced obesity and ameliorates some of the associated metabolic complications. Compared with HFD-fed WT mice, HFD-fed γ-synuclein-null mutant mice display increased lipolysis, lipid oxidation, and energy expenditure, and reduced adipocyte hypertrophy. Knockdown of γ-synuclein in adipocytes causes redistribution of the key lipolytic enzyme ATGL to lipid droplets and increases lipolysis. γ-Synuclein-deficient adipocytes also contain fewer SNARE complexes of a type involved in lipid droplet fusion. We hypothesize that γ-synuclein may deliver SNAP-23 to the SNARE complexes under lipogenic conditions. Via these independent but complementary roles, γ-synuclein may coordinately modulate lipid storage by influencing lipolysis and lipid droplet formation. Our data reveal γ-synuclein as a regulator of lipid handling in adipocytes, the function of which is particularly important in conditions of nutrient excess.

  20. Interaction between -Synuclein and Other Proteins in Neurodegenerative Disorders

    Directory of Open Access Journals (Sweden)

    Kurt A. Jellinger

    2011-01-01

    Full Text Available Protein aggregation is a common characteristic of many neurodegenerative disorders, and the interaction between pathological/toxic proteins to cause neurodegeneration is a hot topic of current neuroscience research. Despite clinical, genetic, and experimental differences, evidence increasingly indicates considerable overlap between synucleinopathies and tauopathies or other protein-misfolding diseases. Inclusions, characteristics of these disorders, also occurring in other neurodegenerative diseases, suggest interactions of pathological proteins engaging common downstream pathways. Novel findings that have shifted our understanding in the role of pathologic proteins in the pathogenesis of Parkinson and Alzheimer diseases have confirmed correlations/overlaps between these and other neurodegenerative disorders. The synergistic effects of α-synuclein, hyperphosphorylated tau, amyloid-β, and other pathologic proteins, and the underlying molecular pathogenic mechanisms, including induction and spread of protein aggregates, are critically reviewed, suggesting a dualism or triad of neurodegeneration in protein-misfolding disorders, although the etiology of most of these processes is still mysterious.

  1. Plasma nitrate clearance in mice: modeling of the systemic production of nitrate following the induction of nitric oxide synthesis.

    Science.gov (United States)

    Veszelovsky, E; Holford, N H; Thomsen, L L; Knowles, R G; Baguley, B C

    1995-01-01

    Nitric oxide (NO) is produced in mammals by the enzyme NO synthase (NOS) in response to a number of agents, including the experimental antitumour agent flavone acetic acid (FAA) and the cytokine tumour necrosis factor-alpha (TNF). NO is converted rapidly in the presence of oxygen, water and haemoglobin to oxidation products, largely nitrate. To quantitate the production of nitric oxide it is necessary to know the clearance of nitrate. The concentration of nitrite and nitrate ion in the plasma of C3H and BDF1 (C57BL6 x DBA2) mice was assessed before and after injection of sodium nitrate and sodium nitrite. Nitrite was covered rapidly to nitrate and the kinetics of elimination of nitrate were determined. There was no significant difference between results obtained with different mouse strains, between levels of nitrite and nitrate, or between i.p. and i.v. administration, and the observations were therefore combined. The volume of distribution of nitrate was 0.71 +/- 0.04 l/kg and the clearance was 0.32 +/- 0.02 l/h-1/kg-1 (plasma half-life, 1.54 h). Using previously published data, we developed a pharmacokinetic-pharmacodynamic model that relates the production of TNF in response to administration of FAA, the enhancement of NOS activity in response to TNF, and the elevation of plasma nitrate in response to NO production. This information permits the prediction from observed plasma nitrate values of the amount of NOS induced in vivo.

  2. α-synuclein genetic variability: A biomarker for dementia in Parkinson disease.

    Science.gov (United States)

    Guella, Ilaria; Evans, Daniel M; Szu-Tu, Chelsea; Nosova, Ekaterina; Bortnick, Stephanie F; Goldman, Jennifer G; Dalrymple-Alford, John C; Geurtsen, Gert J; Litvan, Irene; Ross, Owen A; Middleton, Lefkos T; Parkkinen, Laura; Farrer, Matthew J

    2016-06-01

    The relationship between Parkinson disease (PD), PD with dementia (PDD), and dementia with Lewy bodies (DLB) has long been debated. Although PD is primarily considered a motor disorder, cognitive impairment is often present at diagnosis, and only ∼20% of patients remain cognitively intact in the long term. Alpha-synuclein (SNCA) was first implicated in the pathogenesis of the disease when point mutations and locus multiplications were identified in familial parkinsonism with dementia. In worldwide populations, SNCA genetic variability remains the most reproducible risk factor for idiopathic PD. However, few investigators have looked at SNCA variability in terms of cognitive outcomes. We have used targeted high-throughput sequencing to characterize the 135kb SNCA locus in a large multinational cohort of patients with PD, PDD, and DLB and healthy controls. An analysis of 43 tagging single nucleotide polymorphisms across the SNCA locus shows 2 distinct association profiles for symptoms of parkinsonism and/or dementia, respectively, toward the 3' or the 5' of the SNCA gene. In addition, we define a specific haplotype in intron 4 that is directly associated with PDD. The PDD risk haplotype has been interrogated at single nucleotide resolution and is uniquely tagged by an expanded TTTCn repeat. Our data show that PD, PDD, and DLB, rather than a disease continuum, have distinct genetic etiologies albeit within one genomic locus. Such results may serve as prognostic biomarkers to these disorders, to inform physicians and patients, and to assist in the design and stratification of clinical trials aimed at disease modification. Ann Neurol 2016;79:991-999. © 2016 American Neurological Association.

  3. Nitrate absorption through hydrotalcite reformation.

    Science.gov (United States)

    Frost, Ray L; Musumeci, Anthony W

    2006-10-01

    Thermally activated hydrotalcite based upon a Zn/Al hydrotalcite with carbonate in the interlayer has been used to remove nitrate anions from an aqueous solution resulting in the reformation of a hydrotalcite with a mixture of nitrate and carbonate in the interlayer. X-ray diffraction of the reformed hydrotalcites with a d(003) spacing of 7.60 A shows that the nitrate anion is removed within a 30 min period. Raman spectroscopy shows that two types of nitrate anions exist in the reformed hydrotalcite (a) nitrate bonded to the 'brucite-like' hydrotalcite surface and (b) aquated nitrate anion in the interlayer. Kinetically the nitrate is replaced by the carbonate anion over a 21 h period. Two types of carbonate anions are observed. This research shows that the reformation of a thermally activated hydrotalcite can be used to remove anions such as nitrate from aqueous systems.

  4. Nitrate Leaching Management

    Science.gov (United States)

    Nitrate (NO3) leaching is a significant nitrogen (N) loss process for agriculture that must be managed to minimize NO3 enrichment of groundwater and surface waters. Managing NO3 leaching should involve the application of basic principles of understanding the site’s hydrologic cycle, avoiding excess ...

  5. Expression of γ-synuclein in colorectal cancer tissues and its role on colorectal cancer cell line HCT116

    Institute of Scientific and Technical Information of China (English)

    Qing Ye; Bo Feng; Yuan-Fei Peng; Xue-Hua Chen; Qu Cai; Bei-Qin Yu; Liang-Hui Li; Ming-Yuan Qiu; Bing-Ya Liu; Min-Hua Zheng

    2009-01-01

    AIM: To investigate the expression pattern of γ-synuclein in colorectal cancer (CRC) tissues, and to study the effects of γ-synuclein on CRC cell line HCT116 biological features in vitro.METHODS: The expression pattern of γ-synuclein was determined in 54 CRC tissues and 30 tumormatched nonneoplastic adjacent tissues (NNAT) 5 cm away from the tumor via real-time quantitative reverse transcription PCR (RT-PCR) and immunohistochemistry.The relationship between γ-synuclein protein expression and clinicopathological factors of CRC tissues was analyzed. Three small interfering RNA (siRNA) targeting γ-synuclein mRNA plasmids were constructed and transfected into the CRC cell line HCT116. The stable cell lines were selected with G-418 for 28 d, and the biological features of these cells were examined by cell growth curve, soft agar assay, and cell migration and invasion assays in vitro.RESULTS: The expression of γ-synuclein mRNA and protein was much higher in CRC tissue samples than in NNAT samples ( P = 0.02, P = 0.036). There was a significant correlation between the γ-synuclein protein expression and clinical stage and lymph node involvement of CRC ( P = 0.02, P = 0.033). In functional analysis we found that down-regulation of γ-synuclein expression in HCT116 cells could inhibit the growth, colony formation rate, and migration and invasion ability of HCT116 cells.CONCLUSION: Increased expression of γ-synuclein in CRC tissues and the biological effects of reduced γ-synuclein expression on HCT116 cells suggest that γ-synuclein may play a positive role in the progression of CRC.

  6. Polychlorinated biphenyls alter expression of alpha-synuclein, synaptophysin and parkin in the rat brain

    DEFF Research Database (Denmark)

    Malkiewicz, Katarzyna; Mohammed, Roma; Folkesson, Ronnie

    2006-01-01

    Polychlorinated Biphenyls (PCBs)-induced changes in synaptic transmission are one of the effects of their neurotoxicity but the mechanism remains unknown. We assessed the in vivo effects of the PCBs mixture, Aroclor 1254 on the expression of neuronal proteins that are involved in the synaptic fun...

  7. Αlpha-synuclein levels in blood plasma from LRRK2 mutation carriers.

    Directory of Open Access Journals (Sweden)

    Ana Gorostidi

    Full Text Available The diagnosis of Parkinson's disease (PD remains primarily a clinical issue, based mainly on phenotypic patterns. The identification of biomarkers capable of permitting the preclinical detection of PD is critically needed. α-Synuclein is a key protein in PD, with missense and multiplication mutations in the gene encoding α-synuclein (SNCA having been reported in familial cases of PD, and accumulation of the protein identified in Lewy bodies (LBs and Lewy neurites (LNs in affected brain regions. With the objective of validating the use of α-synuclein as a clinical or progressive biomarker in an accessible tissue, we used an enzyme-linked immunosorbent assay (ELISA to measure α-synuclein levels in the peripheral blood plasma of idiopathic PD and LRRK2 mutation carrier patients and compared our findings with healthy control subjects. Compared to healthy controls, we found a significant decrease in plasma total α-synuclein levels in idiopathic PD (iPD patients (n = 134, p = 0.010. However, the reduction was less significant in patients who were LRRK2 mutation carriers (n = 32, p = 0.133. This lack of significance could be due to the small number of individuals employed in this group. No predictive value of total α-synuclein in the diagnosis of PD was found in a receiver operating characteristic (ROC curve analysis. Although this is a pilot study requiring corroboration on a larger cohort of patients, our results highlight the possible use of plasma α-synuclein as a biomarker for PD.

  8. α-Synuclein in the colon and premotor markers of Parkinson disease in neurologically normal subjects.

    Science.gov (United States)

    Kim, Joong-Seok; Park, In-Seok; Park, Hyung-Eun; Kim, Su-Young; Yun, Jung A; Jung, Chan Kwon; Sung, Hye-Young; Lee, Jin-Kwon; Kang, Won-Kyung

    2017-01-01

    Extranigral non-motor signs precede the first motor manifestations of Parkinson's disease by many years in some patients. The presence of α-synuclein deposition within colon tissues in patients with Parkinson's disease can aid in identifying early neuropathological changes prior to disease onset. In the present study, we evaluated the roles of non-motor symptoms and signs and imaging biomarkers of nigral neuronal changes and α-synuclein accumulation in the colon. Twelve subjects undergoing colectomy for primary colon cancer were recruited for this study. Immunohistochemical staining for α-synuclein in normal and phosphorylated forms was performed in normally appearing colonic tissue. We evaluated 16 candidate premotor risk factors in this study cohort. Among them, ten subjects showed positive immunostaining with normal- and phosphorylated-α-synuclein. An accumulation of premotor markers in each subject was accompanied with positive normal- and phosphorylated-α-synuclein immunostaining, ranging from 2 to 7 markers per subject, whereas the absence of Lewy bodies in the colon was associated with relative low numbers of premotor signs. A principal component analysis and a cluster analysis of these premotor markers suggest that urinary symptoms were commonly clustered with deposition of peripheral phosphorylated-α-synuclein. Among other premotor marker, color vision abnormalities were related to non-smoking. This mathematical approach confirmed the clustering of premotor markers in preclinical stage of Parkinson's disease. This is the first report showing that α-synuclein in the colon and other premotor markers are related to each other in neurologically normal subjects.

  9. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Marie Y Davis

    2016-03-01

    Full Text Available Mutations in the glucosidase, beta, acid (GBA1 gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD and dementia with Lewy bodies (DLB excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity.

  10. Adsorption of α-synuclein to supported lipid bilayers: positioning and role of electrostatics.

    Science.gov (United States)

    Hellstrand, Erik; Grey, Marie; Ainalem, Marie-Louise; Ankner, John; Forsyth, V Trevor; Fragneto, Giovanna; Haertlein, Michael; Dauvergne, Marie-Therese; Nilsson, Hanna; Brundin, Patrik; Linse, Sara; Nylander, Tommy; Sparr, Emma

    2013-10-16

    An amyloid form of the protein α-synuclein is the major component of the intraneuronal inclusions called Lewy bodies, which are the neuropathological hallmark of Parkinson's disease (PD). α-Synuclein is known to associate with anionic lipid membranes, and interactions between aggregating α-synuclein and cellular membranes are thought to be important for PD pathology. We have studied the molecular determinants for adsorption of monomeric α-synuclein to planar model lipid membranes composed of zwitterionic phosphatidylcholine alone or in a mixture with anionic phosphatidylserine (relevant for plasma membranes) or anionic cardiolipin (relevant for mitochondrial membranes). We studied the adsorption of the protein to supported bilayers, the position of the protein within and outside the bilayer, and structural changes in the model membranes using two complementary techniques-quartz crystal microbalance with dissipation monitoring, and neutron reflectometry. We found that the interaction and adsorbed conformation depend on membrane charge, protein charge, and electrostatic screening. The results imply that α-synuclein adsorbs in the headgroup region of anionic lipid bilayers with extensions into the bulk but does not penetrate deeply into or across the hydrophobic acyl chain region. The adsorption to anionic bilayers leads to a small perturbation of the acyl chain packing that is independent of anionic headgroup identity. We also explored the effect of changing the area per headgroup in the lipid bilayer by comparing model systems with different degrees of acyl chain saturation. An increase in area per lipid headgroup leads to an increase in the level of α-synuclein adsorption with a reduced water content in the acyl chain layer. In conclusion, the association of α-synuclein to membranes and its adsorbed conformation are of electrostatic origin, combined with van der Waals interactions, but with a very weak correlation to the molecular structure of the anionic

  11. Crosstalk between the proteasome system and autophagy in the clearance of α-synuclein

    Institute of Scientific and Technical Information of China (English)

    Fang YANG; Ya-ping YANG; Cheng-jie MAO; Ling LIU; Hui-fen ZHENG; Li-fang HU; Chun-feng LIU

    2013-01-01

    A growing body of evidence suggests that α-synuclein accumulation may play an important role in the pathogenesis of Parkinson's disease.The aim of this study was to investigate the roles of the proteasome and autophagy pathways in the clearance of wild-type and mutant α-synuclein in PC12 cells.Methods:PC12 cells overexpressing either wild-type or A30P mutant α-synuclein were treated with the proteasome inhibitor epoxomicin,the macroautophagy inhibitor 3-MA and the macroautophagy activator rapamycin alone or in combination.The cell viability was assessed using MTT assay.Immunofluorescence and Western blot analysis were used to detect the level of α-synuclein,LAMP-2A,E1 activase,and E2 ligase in the cells.Chymotrypsin-like proteasomal activity was measured using a commercial kit.Results:When the proteasome and macroautophagy in the wild-type and mutant cells were inhibited with epoxomicin and 3-MA,respectively,the cell viability was significantly decreased,and the α-synuclein level was increased.Both epoxomicin and 3-MA activated the chaperone-mediated autophagy (CMA) by increasing the level of the CMA-limiting enzyme LAMP-2A.Furthermore,3-MA or epoxomicin significantly decreased chymotrypsin-like proteasomal activity.3-MA or epoxomicin did not change E1 activase expression in either mutant or wild-type cells,but increased E2 ligase expression,especially when used together.Macroautophagy inducer rapamycin increased the cell viability and reduced epoxomicin-induced α-synuclein accumulation.Interestingly,CMA was also activated by rapamycin.Conclusion:Our results demonstrate the existence of complex crosstalk between different forms of autophagy and between autophagy and the proteasome pathway in the clearance of α-synuclein in PC12 cells.

  12. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

    Science.gov (United States)

    Davis, Marie Y; Trinh, Kien; Thomas, Ruth E; Yu, Selina; Germanos, Alexandre A; Whitley, Brittany N; Sardi, Sergio Pablo; Montine, Thomas J; Pallanck, Leo J

    2016-03-01

    Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity.

  13. O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

    Science.gov (United States)

    Marotta, Nicholas P.; Lin, Yu Hsuan; Lewis, Yuka E.; Ambroso, Mark R.; Zaro, Balyn W.; Roth, Maxwell T.; Arnold, Don B.; Langen, Ralf; Pratt, Matthew R.

    2015-11-01

    Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

  14. α-Synuclein binds and sequesters PIKE-L into Lewy bodies, triggering dopaminergic cell death via AMPK hyperactivation.

    Science.gov (United States)

    Kang, Seong Su; Zhang, Zhentao; Liu, Xia; Manfredsson, Fredric P; He, Li; Iuvone, P Michael; Cao, Xuebing; Sun, Yi E; Jin, Lingjing; Ye, Keqiang

    2017-01-31

    The abnormal aggregation of fibrillar α-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson's disease. However, the molecular mechanisms regulating α-synuclein pathological effects are incompletely understood. Here we show that α-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylation-dependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. α-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds α-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP(+)), whereas PIKE-L knockdown elevates α-synuclein oligomerization and cell death. The overexpression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or α-synuclein induces greater dopaminergic cell loss and more severe motor defects in PIKE-KO and Fyn-KO mice than in wild-type mice, and these effects are attenuated by the expression of dominant-negative AMPK. Hence, our findings demonstrate that α-synuclein neutralizes PIKE-L's neuroprotective actions in synucleinopathies, triggering dopaminergic neuronal death by hyperactivating AMPK.

  15. Covalent α-synuclein dimers: chemico-physical and aggregation properties.

    Directory of Open Access Journals (Sweden)

    Micaela Pivato

    Full Text Available The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson's disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1-104 and 29-140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results

  16. 33 CFR 126.28 - Ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate; general provisions. 126.28 Section 126...) WATERFRONT FACILITIES HANDLING OF DANGEROUS CARGO AT WATERFRONT FACILITIES § 126.28 Ammonium...

  17. Bryostatin I inhibits growth of breast cancer cells through the inhibition of synuclein-A expression

    Directory of Open Access Journals (Sweden)

    Jun-Xia Sun

    2016-09-01

    Full Text Available The present study was aimed to investigate the effect of bryostatin I on the expression of synuclein-A in breast cancer cells. Western blot analysis showed a significant (p<0.005 reduction in the expression of synuclein-A from a concentration of 20 µM in H3922 cells. The inhibitory effect of bryostatin I on synuclein-A expression was further confirmed by the treatment of H3922 cells with known synuclein-A inhibitor, cytokine oncostatin M. Bryostatin I treatment of H3922 cells also significantly increased their sensitivity to the taxol. Incubation of the cells with 25 µM concentration of bryostatin I followed by treatment with 0.5 μM concentration of taxol induced apoptosis in 89% cells compared to 9% cells in the taxol alone treated cultures. Treatment of the H3922 cells with bryostatin I at 25 µM concentration led to a significant increase in the activation of histone H1 protein. The results from MTT assay showed a significant decrease in the cell viability from 10 µM concentration of bryostatin I. Thus, bryostatin I inhibits the growth of breast cancer cells through inhibition of synuclein-A expression and can be used for breast cancer treatment.

  18. The yin and yang of α-synuclein-associated epigenetics in Parkinson's disease.

    Science.gov (United States)

    Pavlou, Maria Angeliki S; Pinho, Raquel; Paiva, Isabel; Outeiro, Tiago Fleming

    2017-04-01

    Parkinson's disease is the second most prevalent neurodegenerative disorder. The main neuropathological hallmarks of the disease are the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of protein inclusions known as Lewy bodies. Recently, great attention has been given to the study of genes associated with both familial and sporadic forms of Parkinson's disease. Among them, the α-synuclein gene is believed to play a central role in the disease and is, therefore, one of the most studied genes. Parkinson's disease is a complex disorder and, as such, derives from the interaction between genetic and environmental factors. Here, we offer an update on the landscape of epigenetic-mediated regulation of gene expression that has been linked with α-synuclein and associated with Parkinson's disease. We also provide an overview of how epigenetic modifications can influence the transcription and/or translation of the α-synuclein gene and, on the other hand, how α-synuclein function/dysfunction can, per se, affect the epigenetic landscape. Finally, we discuss how a deeper understanding of the epigenetic profile of α-synuclein may enable the development of novel therapeutic approaches for Parkinson's disease and other synucleinopathies. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Simulation and modeling of synuclein-based 'protofibril' structures: as a means of understanding the molecular basis of Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Tsigelny, I F; Sharikov, Y; Miller, M A; Masliah, E, E-mail: itsigeln@ucsd.edu

    2008-07-15

    The protein {alpha}-synuclein (aS) is a 140 amino acid cytosolic protein of unknown function expressed abundantly in neurons and is associated with both environmentally induced and inherited forms of Parkinson's disease, the neurological disorder caused by progressive death of dopaminergic neurons in the substantia nigra. Here we demonstrate that aS aggregation leads to protofibril formation that accompanies aS binding to membranes. The specific surfaces of aS that bind to membranes are likely developed during the folding maturation process, where the 3D combinations of amino acids that are bound to these surfaces are very specific. Using molecular modeling and simulation technique, we elucidate the folding pathway of aS into protofibril structures and its configurations that bind to membranes. These membrane-binding surfaces can be targeted with pharmaceutical intervention for dissolution.

  20. Nitrate in drinking water

    DEFF Research Database (Denmark)

    Schullehner, Jörg; Hansen, Birgitte; Sigsgaard, Torben

    Annual nationwide exposure maps for nitrate in drinking water in Denmark from the 1970s until today will be presented based on the findings in Schullehner & Hansen (2014) and additional work on addressing the issue of private well users and estimating missing data. Drinking water supply in Denmark...... is highly decentralized and fully relying on simple treated groundwater. At the same time, Denmark has an intensive agriculture, making groundwater resources prone to nitrate pollution. Drinking water quality data covering the entire country for over 35 years are registered in the public database Jupiter....... In order to create annual maps of drinking water quality, these data had to be linked to 2,852 water supply areas, which were for the first time digitized, collected in one dataset and connected to the Jupiter database. Analyses of the drinking water quality maps showed that public water supplies...

  1. Alpha Blockers

    Science.gov (United States)

    ... positive side, alpha blockers might decrease low-density lipoprotein (LDL) cholesterol (the "bad" cholesterol). Alpha blockers can ... PreventionTreatmentofHighBloodPressure/Types-of-Blood-Pressure-Medications_UCM_303247_Article.jsp. Accessed June 4, 2016. Kaplan NM, et ...

  2. Design, Synthesis, and Characterization of 3-(Benzylidene)indolin-2-one Derivatives as Ligands for α-Synuclein Fibrils.

    Science.gov (United States)

    Chu, Wenhua; Zhou, Dong; Gaba, Vrinda; Liu, Jialu; Li, Shihong; Peng, Xin; Xu, Jinbin; Dhavale, Dhruva; Bagchi, Devika P; d'Avignon, André; Shakerdge, Naomi B; Bacskai, Brian J; Tu, Zhude; Kotzbauer, Paul T; Mach, Robert H

    2015-08-13

    A series of 3-(benzylidine)indolin-2-one derivatives were synthesized and evaluated for their in vitro binding to alpha synuclein (α-syn), beta amyloid (Aβ), and tau fibrils. Compounds with a single double bond in the 3-position had only a modest affinity for α-syn and no selectivity for α-syn versus Aβ or tau fibrils. Homologation to the corresponding diene analogues yielded a mixture of Z,E and E,E isomers; substitution of the indoline nitrogen with an N-benzyl group resulted in increased binding to α-syn and reasonable selectivity for α-syn versus Aβ and tau. Introduction of a para-nitro group into the benzene ring of the diene enabled separation of the Z,E and E,E isomers and led to the identification of the Z,E configuration as the more active regioisomer. The data described here provide key structural information in the design of probes which bind preferentially to α-syn versus Aβ or tau fibrils.

  3. Methyl-branched lipids promote the membrane adsorption of α-synuclein by enhancing shallow lipid-packing defects.

    Science.gov (United States)

    Garten, Matthias; Prévost, Coline; Cadart, Clotilde; Gautier, Romain; Bousset, Luc; Melki, Ronald; Bassereau, Patricia; Vanni, Stefano

    2015-06-28

    Alpha-synuclein (AS) is a synaptic protein that is directly involved in Parkinson's disease due to its tendency to form protein aggregates. Since AS aggregation can be dependent on the interactions between the protein and the cell plasma membrane, elucidating the membrane binding properties of AS is of crucial importance to establish the molecular basis of AS aggregation into toxic fibrils. Using a combination of in vitro reconstitution experiments based on Giant Unilamellar Vesicles (GUVs), confocal microscopy and all-atom molecular dynamics simulations, we have investigated the membrane binding properties of AS, with a focus on the relative contribution of hydrophobic versus electrostatic interactions. In contrast with previous observations, we did not observe any binding of AS to membranes containing the ganglioside GM1, even at relatively high GM1 content. AS, on the other hand, showed a stronger affinity for neutral flat membranes consisting of methyl-branched lipids. To rationalize these results, we used all-atom molecular dynamics simulations to investigate the influence of methyl-branched lipids on interfacial membrane properties. We found that methyl-branched lipids promote the membrane adsorption of AS by creating shallow lipid-packing defects to a larger extent than polyunsaturated and monounsaturated lipids. Our findings suggest that methyl-branched lipids may constitute a remarkably adhesive substrate for peripheral proteins that adsorb on membranes via hydrophobic insertions.

  4. Cyclodextrins 3-Functionalized with 8-Hydroxyquinolines: Copper-Binding Ability and Inhibition of Synuclein Aggregation.

    Science.gov (United States)

    Oliveri, Valentina; Sgarlata, Carmelo; Vecchio, Graziella

    2016-09-06

    Neurodegenerative diseases such as Parkinson's and Alzheimer's diseases are multifactorial disorders related to protein aggregation, metal dyshomeostasis, and oxidative stress. To advance understanding in this area and to contribute to therapeutic development, many efforts have been directed at devising suitable agents that can target metal ions associated with relevant biomolecules such as α-synuclein. This paper presents a new cyclodextrin-8-hydroxyquinoline conjugate and discusses the properties of four cyclodextrins 3-functionalized with 8-hydroxyquinoline as copper(II) chelators and inhibitors of copper-induced synuclein aggregation. The encouraging results establish the potential of cyclodextrin-8-hydroxyquinoline conjugates as chelators for the control of copper toxicity.

  5. Assimilation of nitrate by yeasts.

    Science.gov (United States)

    Siverio, José M

    2002-08-01

    Nitrate assimilation has received much attention in filamentous fungi and plants but not so much in yeasts. Recently the availability of classical genetic and molecular biology tools for the yeast Hansenula polymorpha has allowed the advance of the study of this metabolic pathway in yeasts. The genes YNT1, YNR1 and YNI1, encoding respectively nitrate transport, nitrate reductase and nitrite reductase, have been cloned, as well as two other genes encoding transcriptional regulatory factors. All these genes lie closely together in a cluster. Transcriptional regulation is the main regulatory mechanism that controls the levels of the enzymes involved in nitrate metabolism although other mechanisms may also be operative. The process involved in the sensing and signalling of the presence of nitrate in the medium is not well understood. In this article the current state of the studies of nitrate assimilation in yeasts as well as possible venues for future research are reviewed.

  6. α-Synuclein potentiates interleukin-1β-induced CXCL10 expression in human A172 astrocytoma cells.

    Science.gov (United States)

    Tousi, Neda Saffarian; Buck, Daniel J; Curtis, J Thomas; Davis, Randall L

    2012-01-24

    Neuroinflammation and neuronal degeneration observed in Parkinson's disease (PD) has been attributed in part to glial-mediated events. Increased expression of proinflammatory cytokines and abnormal accumulation of the neuronal protein, α-synuclein in the brain are also characteristic of PD. While increasing evidence suggests that astrocytes contribute to neuroinflammation and dopaminergic neuronal degeneration associated with PD, there remains much to learn about these astroglial-mediated events. Therefore, we investigated the in vitro effects of interleukin-1β (IL-1β) and α-synuclein on astroglial expression of interferon-γ inducible protein-10 (CXCL10), a proinflammatory and neurotoxic chemokine. IL-1β-induced CXCL10 protein expression was potentiated by co-exposure to α-synuclein. α-Synuclein did not significantly affect IL-1β-induced CXCL10 mRNA expression, but did mediate increased CXCL10 mRNA stability, which may explain, in part, the increased levels of secreted CXCL10 protein. Future investigations are warranted to more fully define the mechanism by which α-synuclein enhances IL-1β-induced astroglial CXCL10 expression. These findings highlight the importance of α-synuclein in modulating inflammatory events in astroglia. These events may be particularly relevant to the pathology of CNS disorders involving α-synuclein accumulation, including PD and HIV-1 associated dementia.

  7. Alpha fetoprotein

    Science.gov (United States)

    Fetal alpha globulin; AFP ... Greater than normal levels of AFP may be due to: Cancer in testes , ovaries, biliary (liver secretion) tract, stomach, or pancreas Cirrhosis of the liver Liver cancer ...

  8. Inhibition of α-Synuclein Fibril Elongation by Hsp70 Is Governed by a Kinetic Binding Competition between α-Synuclein Species.

    Science.gov (United States)

    Aprile, Francesco A; Arosio, Paolo; Fusco, Giuliana; Chen, Serene W; Kumita, Janet R; Dhulesia, Anne; Tortora, Paolo; Knowles, Tuomas P J; Vendruscolo, Michele; Dobson, Christopher M; Cremades, Nunilo

    2017-03-07

    The Hsp70 family of chaperones plays an essential role in suppressing protein aggregation in the cell. Here we investigate the factors controlling the intrinsic ability of human Hsp70 to inhibit the elongation of amyloid fibrils formed by the Parkinson's disease-related protein α-synuclein. Using kinetic analysis, we show that Hsp70 binds preferentially to α-synuclein fibrils as a consequence of variations in the association and dissociation rate constants of binding to the different aggregated states of the protein. Our findings illustrate the importance of the kinetics of binding of molecular chaperones, and also of potential therapeutic molecules, in the efficient suppression of specific pathogenic events linked to neurodegeneration.

  9. Nitrate storage and dissimilatory nitrate reduction by eukaryotic microbes

    DEFF Research Database (Denmark)

    Kamp, Anja; Høgslund, Signe; Risgaard-Petersen, Nils;

    2015-01-01

    The microbial nitrogen cycle is one of the most complex and environmentally important element cycles on Earth and has long been thought to be mediated exclusively by prokaryotic microbes. Rather recently, it was discovered that certain eukaryotic microbes are able to store nitrate intracellularly...... and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate...... storage and dissimilatory nitrate reduction by diverse marine eukaryotes placed into an eco-physiological context. The advantage of intracellular nitrate storage for anaerobic energy conservation in oxygen-depleted habitats is explained and the life style enabled by this metabolic trait is described...

  10. FTIR spectroscopy combined with quantum chemical calculations to investigate adsorbed nitrate on aluminium oxide surfaces in the presence and absence of co-adsorbed water.

    Science.gov (United States)

    Baltrusaitis, Jonas; Schuttlefield, Jennifer; Jensen, Jan H; Grassian, Vicki H

    2007-09-28

    Surface reactions of nitrogen oxides with aluminium oxide particles result in the formation of adsorbed nitrate. Specifically, when alpha-Al(2)O(3) and gamma-Al(2)O(3) particles are exposed to gas-phase NO(2) and HNO(3) adsorbed nitrate forms on the surface. In this study, Fourier transform infrared (FTIR) spectroscopy is combined with quantum chemical calculations to further our understanding of the adsorbed nitrate product on aluminium oxide particle surfaces in the presence and absence of co-adsorbed water at 296 K. FTIR spectra of adsorbed nitrate on alpha-Al(2)O(3) and gamma-Al(2)O(3) particles are interpreted using calculated vibrational frequencies of nitrate coordinated to binuclear Al oxide cluster models. Comparison of the calculated and experimental vibrational frequencies of adsorbed nitrate establishes different modes of coordination (monodentate, bidentate and bridging) of the nitrate ion to the surface in the absence of adsorbed water. In the presence of co-adsorbed water, the nitrate ion becomes fully solvated, as shown by a comparison of the experimental nitrate infrared spectra as a function of relative humidity with the calculated nitrate vibrational frequencies for binuclear Al cluster compounds which contain both coordinated nitrate ions and water molecules. These calculations also suggest that adsorbed water can displace nitrate from direct coordination to the surface, leading to an outer-sphere nitrate adsorption complex as well as an inner-sphere complex. Furthermore, the relative humidity dependence of the spectra suggest that water does not evenly wet the surface even at high relative humidity, as there are open or bare surface sites where nitrate ions are not solvated. Besides adsorbed mondendate, bidendate, bridging and solvated nitrate, the presence of ion bound nitrate ion, partially solvated nitrate, molecular nitric acid, hydronium ion and H(3)O(+):NO(3)(-) ion pairs on the oxide surface are also discussed.

  11. Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy.

    Science.gov (United States)

    Levin, Johannes; Hillmer, Andreas S; Högen, Tobias; McLean, Pamela J; Giese, Armin

    2016-08-12

    Synucleinopathies such as dementia with Lewy bodies or Parkinson's disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Mechanistic study of the inhibitory activity of Geum urbanum extract against α-Synuclein fibrillation

    DEFF Research Database (Denmark)

    Lobbens, Eva Stephanie; Breydo, Leonid; Pedersen, Thomas Skamris

    2016-01-01

    The presence of Lewy bodies and Lewy neurites is a major pathological hallmark of Parkinson's disease and is hypothesized to be linked to disease development, although this is not yet conclusive. Lewy bodies and Lewy neurites primarily consist of fibrillated α-Synuclein; yet, there is no treatmen...

  13. α-Synuclein and DJ-1 as potential biological fluid biomarkers for Parkinson's Disease.

    Science.gov (United States)

    Waragai, Masaaki; Sekiyama, Kazunari; Sekigawa, Akio; Takamatsu, Yoshiki; Fujita, Masayo; Hashimoto, Makoto

    2010-10-29

    Parkinson's disease (PD) is the most common form of movement disorder and affects approximately 4% of the population aged over 80 years old. Currently, PD cannot be prevented or cured, and no single diagnostic biomarkers are available. Notably, recent studies suggest that two familial PD-linked molecules, α-synuclein and DJ-1, are present in cerebrospinal fluid (CSF) and that their levels may be altered during the progression of PD. In this regard, sensitive and accurate methods for evaluation of α-synuclein and DJ-1 levels in the CSF and blood have been developed, and the results suggest that the levels of both molecules are significantly decreased in the CSF in patients with PD compared with age-matched controls. Furthermore, specific detection and quantification of neurotoxic oligometric forms of α-synuclein in the blood using enzyme-linked immunosorbent assays might be expected as potential peripheral biomarkers for PD, although further validation is required. Currently, neither α-synuclein nor DJ-1 is satisfactory as a single biomarker for PD, but combinatory evaluation of these biological fluid molecules with other biomarkers and imaging techniques may provide reliable information for diagnosis of PD.

  14. Decreasing α-synuclein aggregation by methanolic extract of Centella asiatica in zebrafish Parkinson's model

    Directory of Open Access Journals (Sweden)

    Husnul Khotimah

    2015-11-01

    Conclusions: C. asiatica has a potential to be developed as an anti-Parkinson's disease treatment due to its capability for minimized the sign of Parkinson's such as α-synuclein aggregation and expression, increasing motility and dopamine as well.

  15. Insights into the function and dysfunction of α-synuclein in cells

    NARCIS (Netherlands)

    Raiss, Christian Carl-Michael

    2015-01-01

    This thesis sheds light on the function and dysfunction of the protein α-synuclein (α-S) in the test tube and in cells and ultimately its possible involvement in Parkinson’s disease (PD). Following the introduction in Chapter 1, Chapters 2 and 3 concentrate on the investigation of the interaction be

  16. Chameleon behaviour of α-synuclein: brownian dynamics simulations of protein aggregation

    NARCIS (Netherlands)

    Ilie, Ioana-Mariuca

    2015-01-01

    Over the past decades a large number of studies have been carried out in order to determine the physiological function of α-synuclein and its implication in Parkinson's disease. A complementary tool to experiments are computer simulations, which are intensively used for problems for which experiment

  17. The role of stable α-synuclein oligomers in the molecular events underlying amyloid formation

    DEFF Research Database (Denmark)

    Lorenzen, Nikolai; Nielsen, Søren Bang; Buell, Alexander K.

    2014-01-01

    α-synuclein (αSN), whose aggregation is strongly implicated in the development of Parkinson’s disease (PD). The two types of oligomers are both formed under conditions where amyloid fibril formation is observed but differ in molecular weight by an order of magnitude. Both possess a degree of β...

  18. TMEM175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation

    Science.gov (United States)

    Jinn, Sarah; Drolet, Robert E.; Cramer, Paige E.; Wong, Andus Hon-Kit; Toolan, Dawn M.; Gretzula, Cheryl A.; Voleti, Bhavya; Vassileva, Galya; Disa, Jyoti; Tadin-Strapps, Marija; Stone, David J.

    2017-01-01

    Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD. PMID:28193887

  19. Specific fluorescent detection of fibrillar α-synuclein using mono- and trimethine cyanine dyes

    NARCIS (Netherlands)

    Volkova, K.D.; Kovalska, V.B.; Balanda, A.O.; Losytskyy, M.Yu; Golub, A.G.; Vermeij, R.J.; Subramaniam, V.; Tolmachev, O.I.; Yarmoluk, S.M.

    2008-01-01

    With the aim of searching of novel amyloid-specific fluorescent probes the ability of series of mono- and trimethine cyanines based on benzothiazole, pyridine and quinoline heterocycle end groups to recognize fibrillar formations of α-synuclein (ASN) was studied. For the first time it was revealed t

  20. Copper(II) enhances membrane-bound α-synuclein helix formation.

    Science.gov (United States)

    Lucas, Heather R; Lee, Jennifer C

    2011-03-01

    Interactions of copper and membranes with α-synuclein have been implicated in pathogenic mechanisms of Parkinson's disease, yet work examining both concurrently is scarce. We have examined the effect of copper(ii) on protein/vesicle binding and found that both the copper(ii) affinity and α-helical content are enhanced for the membrane-bound protein.

  1. Copper(II) enhances membrane-bound α-synuclein helix formation

    OpenAIRE

    Lucas, Heather R.; Lee, Jennifer C.

    2011-01-01

    Interactions of copper and membranes with α-synuclein have been implicated in pathogenic mechanisms of Parkinson’s disease, yet work examining both concurrently is scarce. We have examined the effect of copper(II) on protein/vesicle binding and found that both the copper(II) affinity and α-helical content are enhanced for the membrane-bound protein.

  2. Structural and dynamical insights into the membrane-bound α-synuclein.

    Directory of Open Access Journals (Sweden)

    Neha Jain

    Full Text Available Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson's disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES, which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could

  3. Waterproofing Materials for Ammonium Nitrate

    Directory of Open Access Journals (Sweden)

    R.S. Damse

    2004-10-01

    Full Text Available This study explores the possibility of overcoming the problem of hygroscopicity of ammonium nitrate by coating the particles with selected waterproofing materials. Gravimetric analysis ofthe samples of ammonium nitrate coated with eight different waterproofing materials, vis-a-vis, uncoated ammonium nitrate, were conducted at different relative humidity and exposuretime. The results indicate that mineral jelly is the promising waterproofing material for ammonium nitrate among the materials tested, viz, calcium stearate, dioctyl phthalate, kaoline, diethylphthalate, dinitrotoluene, shelac varnish, and beeswax. Attempts were made to confirm the waterproofing ability of mineral jelly to ammonium nitrate using differential thermal analysisand x-ray diffraction patterns as an experimental tool. Suitability of mineral jelly as an additive for the gun propellant was also assessed on the basis of theoretical calculations using THERMprogram.

  4. Nitration of Polystyrene-Part II Effect of Nitrating Medium on Nitration

    Directory of Open Access Journals (Sweden)

    I. Bajaj

    1968-04-01

    Full Text Available Polystyrene has been nitrated in mixtures of anhydrous nitric and sulphuric acid (70 : 30 and 80 : 20 by volume. Degree of substitution of nitro group per benzene ring varies from 1 to 2 depending on the time, temperature and composition of the nitrating media. Effect of polar and non polar solvents on nitration has been studied by nitrating the polymer in (i fuming nitric acid and (iimixture of nitric and sulphuric acid in presence of dimethyl formamide (DMF and carbon tetrachloride (CCI/Sub4. MF increase the rate of nitration in fuming nitric acid whereas the rate of nitration is lowered in the presence of DMF in the nitrating mixtures. In the case of CCI/Sub4, however, the effect is just the opposite to that observed in DMF. The results have been explained from the mechanism of the formation of 'nitroniumion,NO/Sub2+ in various nitrating media. Degradation of the polymer has been found to be comparatively less in the presence of the organic solvents used in the study.

  5. Mitochondrial Dysfunction and α-Synuclein Synaptic Pathology in Parkinson’s Disease: Who’s on First?

    Directory of Open Access Journals (Sweden)

    Michela Zaltieri

    2015-01-01

    Full Text Available Parkinson’s disease (PD is the most common neurodegenerative movement disorder. Its characteristic neuropathological features encompass the loss of dopaminergic neurons of the nigrostriatal system and the presence of Lewy bodies and Lewy neurites. These are intraneuronal and intraneuritic proteinaceous insoluble aggregates whose main constituent is the synaptic protein α-synuclein. Compelling lines of evidence indicate that mitochondrial dysfunction and α-synuclein synaptic deposition may play a primary role in the onset of this disorder. However, it is not yet clear which of these events may come first in the sequel of processes leading to neurodegeneration. Here, we reviewed data supporting either that α-synuclein synaptic deposition precedes and indirectly triggers mitochondrial damage or that mitochondrial deficits lead to neuronal dysfunction and α-synuclein synaptic accumulation. The present overview shows that it is still difficult to establish the exact temporal sequence and contribution of these events to PD.

  6. $\\alpha_s$ review (2016)

    CERN Document Server

    d'Enterria, David

    2016-01-01

    The current world-average of the strong coupling at the Z pole mass, $\\alpha_s(m^2_{Z}) = 0.1181 \\pm 0.0013$, is obtained from a comparison of perturbative QCD calculations computed, at least, at next-to-next-to-leading-order accuracy, to a set of 6 groups of experimental observables: (i) lattice QCD "data", (ii) $\\tau$ hadronic decays, (iii) proton structure functions, (iv) event shapes and jet rates in $e^+e^-$ collisions, (v) Z boson hadronic decays, and (vi) top-quark cross sections in p-p collisions. In addition, at least 8 other $\\alpha_s$ extractions, usually with a lower level of theoretical and/or experimental precision today, have been proposed: pion, $\\Upsilon$, W hadronic decays; soft and hard fragmentation functions; jets cross sections in pp, e-p and $\\gamma$-p collisions; and photon F$_2$ structure function in $\\gamma\\,\\gamma$ collisions. These 14 $\\alpha_s$ determinations are reviewed, and the perspectives of reduction of their present uncertainties are discussed.

  7. Urea and thiourea modified polypropyleneimine dendrimers clear intracellular α-synuclein aggregates in a human cell line

    DEFF Research Database (Denmark)

    Laumann, Kristoffer; Boas, Ulrik; Larsen, Hjalte Martin;

    2015-01-01

    Synucleinopathies are neurodegenerative pathologies in which disease progression is closely correlated to brain accumulation of insoluble α-synuclein, a small protein abundantly expressed in neural tissue. Here, two types of modified polypropyleneimine (PPI) dendrimers having either urea or methy......Synucleinopathies are neurodegenerative pathologies in which disease progression is closely correlated to brain accumulation of insoluble α-synuclein, a small protein abundantly expressed in neural tissue. Here, two types of modified polypropyleneimine (PPI) dendrimers having either urea...

  8. Prolongation of chemically-induced methemoglobinemia in mice lacking α-synuclein: A novel pharmacologic and toxicologic phenotype

    Directory of Open Access Journals (Sweden)

    Yien-Ming Kuo

    2015-01-01

    Full Text Available The protein α-synuclein is considered central to the pathogenesis of Parkinson disease (PD on genetic and histopathological grounds. It is widely expressed in fetal life and continues to be highly expressed in adult neural tissues, red blood cells and platelets, while the remainder of adult tissues are reported to have little or no expression. Despite cellular and molecular evidence for a role in neuronal function including synaptic vesicle trafficking, neurotransmitter release, mitochondrial function, lipid metabolism, neurogenesis, neuroprotection, and neuromelanin biosynthesis, mice ablated for the gene encoding α-synuclein (Snca have little or no neurological phenotype. Thus, nearly 20 years of intensive study have yet to reveal conclusively what the normal function of this highly abundant protein is in the nervous system. Interestingly, α-synuclein has also been shown to have enzymatic activity as a ferrireductase capable of reducing Fe+3 to Fe+2. Given its abundant expression in red blood cells, we set out to explore the role of α-synuclein in converting chemically-induced Fe+3 methemoglobin to normal Fe+2 hemoglobin. Initial in vivo experiments with the potent methemoglobin inducer, para-aminopropiophenone and its active metabolite, 4-hydroxy para-aminopropiophenone, demonstrated significantly greater and more prolonged methemoglobinemia in Snca−/− mice compared to Snca+/+ mice. In vitro experiments with red blood cells, however, and in vivo experiments in genetically engineered mouse strains that differ in their α-synuclein expression in various tissues, including the nervous system, red blood cells and liver, revealed that contrary to the initial hypothesis, a lack of expression of α-synuclein in red blood cells did not correlate with higher levels or more prolonged duration of methemoglobinemia. Instead, the greater sensitivity to chemically induced methemoglobinemia correlated with the absence of hepatic α-synuclein

  9. α-Synuclein Over-Expression Induces Increased Iron Accumulation and Redistribution in Iron-Exposed Neurons.

    Science.gov (United States)

    Ortega, Richard; Carmona, Asuncion; Roudeau, Stéphane; Perrin, Laura; Dučić, Tanja; Carboni, Eleonora; Bohic, Sylvain; Cloetens, Peter; Lingor, Paul

    2016-04-01

    Parkinson's disease is the most common α-synucleinopathy, and increased levels of iron are found in the substantia nigra of Parkinson's disease patients, but the potential interlink between both molecular changes has not been fully understood. Metal to protein binding assays have shown that α-synuclein can bind iron in vitro; therefore, we hypothesized that iron content and iron distribution could be modified in cellulo, in cells over-expressing α-synuclein. Owing to particle-induced X-ray emission and synchrotron X-ray fluorescence chemical nano-imaging, we were able to quantify and describe the iron distribution at the subcellular level. We show that, in neurons exposed to excess iron, the mere over-expression of human α-synuclein results in increased levels of intracellular iron and in iron redistribution from the cytoplasm to the perinuclear region within α-synuclein-rich inclusions. Reproducible results were obtained in two distinct recombinant expression systems, in primary rat midbrain neurons and in a rat neuroblastic cell line (PC12), both infected with viral vectors expressing human α-synuclein. Our results link two characteristic molecular features found in Parkinson's disease, the accumulation of α-synuclein and the increased levels of iron in the substantia nigra.

  10. Dopamine-mediated oxidation of methionine 127 in α-synuclein causes cytotoxicity and oligomerization of α-synuclein.

    Directory of Open Access Journals (Sweden)

    Kazuhiro Nakaso

    Full Text Available Parkinson's disease (PD is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. Many recent studies focused on the interaction between α-synuclein (α-syn and dopamine in the pathogenesis of PD, and fluorescent anisotropy suggested that the C-terminal region of α-syn may be a target for modification by dopamine. However, it is not well understood why PD-related pathogenesis occurs selectively in dopaminergic neurons. We investigated the interaction between dopamine and α-syn with regard to cytotoxicity. A soluble oligomer was formed by co-incubating α-syn and dopamine in vitro. To clarify the effect of dopamine on α-syn in cells, we generated PC12 cells expressing human α-syn, as well as the α-syn mutants, M116A, Y125D, M127A, S129A, and M116A/M127A, in a tetracycline-inducible manner (PC12-TetOFF-α-syn. Overexpression of wildtype α-syn in catecholaminergic PC12 cells decreased cell viability in long-term cultures, while a competitive inhibitor of tyrosine hydroxylase blocked this vulnerability, suggesting that α-syn-related cytotoxicity is associated with dopamine metabolism. The vulnerabilities of all mutant cell lines were lower than that of wildtype α-syn-expressing cells. Moreover, α-syn containing dopamine-mediated oxidized methionine (Met(O was detected in PC12-TetOFF-α-syn. Met(O was lower in methionine mutant cells, especially in the M127A or M116A/M127A mutants, but also in the Y125D and S129A mutants. Co-incubation of dopamine and the 125YEMPS129 peptide enhanced the production of H2O2, which may oxidize methionine residues and convert them to Met(O. Y125- or S129-lacking peptides did not enhance the dopamine-related production of H2O2. Our results suggest that M127 is the major target for oxidative modification by dopamine, and that Y125 and S129 may act as enhancers of this modification. These results may describe a mechanism of dopaminergic neuron

  11. Light-induced degradation of storage starch in turions of Spirodela polyrhiza depends on nitrate.

    Science.gov (United States)

    Appenroth, Klaus-J; Ziegler, Paul

    2008-10-01

    Light induces both the germination of turions of the duckweed Spirodela polyrhiza and the degradation of the reserve starch stored in the turions. The germination photoresponse requires nitrate, and we show here that nitrate is also needed for the light-induced degradation of the turion starch. Ammonium cannot substitute for nitrate in this regard, and nitrate thus acts specifically as signal to promote starch degradation in the turions. Irradiation with continuous red light leads to starch degradation via auto-phosphorylation of starch-associated glucan, water dikinase (GWD), phosphorylation of the turion starch and enhanced binding of alpha-amylase to starch granules. The present study shows that all of these processes require the presence of nitrate, and that nitrate exerts its effect on starch degradation at a point between the absorption of light by phytochrome and the auto-phosphorylation of the GWD. Nitrate acts to coordinate carbon and nitrogen metabolism in germinating turions: starch will only be broken down when sufficient nitrogen is present to ensure appropriate utilization of the released carbohydrate. These data constitute the first report of control over the initiation of reserve starch degradation by nitrate.

  12. Variability of nitrate and phosphate

    Digital Repository Service at National Institute of Oceanography (India)

    Sardessai, S.; Sundar, D.

    Nitrate and phosphate are important elements of the biogeochemical system of an estuary. Observations carried out during the dry season April-May 2002, and March 2003 and wet season September 2002, show temporal and spatial variability of these two...

  13. Nitrate Reductase: Properties and Regulation

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Nitrate Reductase (NR) is a rating-limit and key enzyme of nitrate assimilation in plants ,so ,NR activity is important for growth,development and the dry matter accumulation of plants. The regulation of NR activity appears to be rather complex and many studies have been devoted to the description of regulation and properties,but in this paper we focus on the properties and regulation of NR in higher plants.

  14. Headspace Analysis of Ammonium Nitrate

    Science.gov (United States)

    2017-01-25

    Naval Research Laboratory Washington, DC 20375-5320 NRL/MR/6110--17-9709 Headspace Analysis of Ammonium Nitrate January 25, 2017 Approved for public...TELEPHONE NUMBER (include area code) b. ABSTRACT c. THIS PAGE 18. NUMBER OF PAGES 17. LIMITATION OF ABSTRACT Headspace Analysis of Ammonium Nitrate G...isobutane reagent ion from analysis of ammonia desorbed from packed tungsten oxide sampling tube .................. 18 E-1 Executive Summary The

  15. 21 CFR 172.170 - Sodium nitrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium nitrate. 172.170 Section 172.170 Food and... Preservatives § 172.170 Sodium nitrate. The food additive sodium nitrate may be safely used in or on specified... sablefish, smoked, cured salmon, and smoked, cured shad, so that the level of sodium nitrate does not...

  16. Nitrate transport and signalling.

    Science.gov (United States)

    Miller, Anthony J; Fan, Xiaorong; Orsel, Mathilde; Smith, Susan J; Wells, Darren M

    2007-01-01

    Physiological measurements of nitrate (NO(3)(-)) uptake by roots have defined two systems of high and low affinity uptake. In Arabidopsis, genes encoding both of these two uptake systems have been identified. Most is known about the high affinity transport system (HATS) and its regulation and yet measurements of soil NO(3)(-) show that it is more often available in the low affinity range above 1 mM concentration. Several different regulatory mechanisms have been identified for AtNRT2.1, one of the membrane transporters encoding HATS; these include feedback regulation of expression, a second component protein requirement for membrane targeting and phosphorylation, possibly leading to degradation of the protein. These various changes in the protein may be important for a second function in sensing NO(3)(-) availability at the surface of the root. Another transporter protein, AtNRT1.1 also has a role in NO(3)(-) sensing that, like AtNRT2.1, is independent of their transport function. From the range of concentrations present in the soil it is proposed that the NO(3)(-)-inducible part of HATS functions chiefly as a sensor for root NO(3)(-) availability. Two other key NO(3)(-) transport steps for efficient nitrogen use by crops, efflux across membranes and vacuolar storage and remobilization, are discussed. Genes encoding vacuolar transporters have been isolated and these are important for manipulating storage pools in crops, but the efflux system is yet to be identified. Consideration is given to how well our molecular and physiological knowledge can be integrated as well to some key questions and opportunities for the future.

  17. Porcine gamma-synuclein: molecular cloning, expression analysis, chromosomal localization and functional expression

    DEFF Research Database (Denmark)

    Frandsen, Pernille Munk; Madsen, Lone Bruhn; Bendixen, Christian

    2009-01-01

    which shows a high similarity to bovine (90%), human (87%) and mouse (83%) γ-synuclein. A genomic clone containing the entire porcine SNCG gene was isolated and its genomic organization determined. The gene is composed of five exons, the general structure being observed to be very similar...... reports the cloning and characterization of the porcine (Sus scrofa) γ-synuclein cDNA (SNCG). The SNCG cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine SNCG cDNA codes for a protein of 126 amino acids...... to that of the human SNCG gene. Expression analysis by quantitative real-time RT-PCR revealed the presence of SNCG transcripts in all examined organs and tissues. Differential expression was observed, with very high levels of SNCG mRNA in fat tissue and high expression levels in spleen, cerebellum, frontal cortex...

  18. Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Levin, Johannes [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich (Germany); Hillmer, Andreas S. [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany); Högen, Tobias [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); McLean, Pamela J. [Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224 (United States); Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany)

    2016-08-12

    Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. - Highlights: • Single particle spectroscopy detects intracellular formed α-synuclein aggregates. • Fusion proteins allow detection of protein

  19. Effects of baseline CSF α-synuclein on regional brain atrophy rates in healthy elders, mild cognitive impairment and Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Niklas Mattsson

    Full Text Available BACKGROUND: Cerebrospinal fluid (CSF α-synuclein is reduced in synucleinopathies, including dementia with Lewy bodies, and some studies have found increased CSF α-synuclein in Alzheimer's disease (AD. No study has explored effects of CSF α-synuclein on brain atrophy. Here we tested if baseline CSF α-synuclein affects brain atrophy rates and if these effects vary across brain regions, and across the cognitive spectrum from healthy elders (NL, to patients with mild cognitive impairment (MCI and AD. METHODS: Baseline CSF α-synuclein measurements and longitudinal structural brain magnetic resonance imaging was performed in 74 NL, 118 MCI patients and 55 AD patients. Effects of baseline CSF α-synuclein on regional atrophy rates were tested in 1 four pre-hoc defined regions possibly associated with Lewy body and/or AD pathology (amygdala, caudate, hippocampus, brainstem, and 2 all available regions of interest. Differences across diagnoses were tested by assessing the interaction of CSF α-synuclein and diagnosis (testing NL versus MCI, and NL versus AD. RESULTS: The effects of CSF α-synuclein on longitudinal atrophy rates were not significant after correction for multiple comparisons. There were tendencies for effects in AD in caudate (higher atrophy rates in subjects with higher CSF α-synuclein, P=0.046 and brainstem (higher atrophy rates in subjects with lower CSF α-synuclein, P=0.063. CSF α-synuclein had significantly different effects on atrophy rates in NL and AD in brainstem (P=0.037 and caudate (P=0.006. DISCUSSION: With the possible exception of caudate and brainstem, the overall weak effects of CSF α-synuclein on atrophy rates in NL, MCI and AD argues against CSF α-synuclein as a biomarker related to longitudinal brain atrophy in these diagnostic groups. Any effects of CSF α-synuclein may be attenuated by possible simultaneous occurrence of AD-related neuronal injury and concomitant Lewy body pathology, which may elevate and

  20. α-synuclein reactive antibodies as diagnostic biomarkers in blood sera of Parkinson's disease patients.

    Directory of Open Access Journals (Sweden)

    Kiran Yanamandra

    Full Text Available BACKGROUND: Auto-antibodies with specificity to self-antigens have been implicated in a wide variety of neurological diseases, including Parkinson's (PD and Alzheimer's diseases, being sensitive indicators of neurodegeneration and focus for disease prevention. Of particular interest are the studies focused on the auto-immune responses to amyloidogenic proteins associated with diseases and their applications in therapeutic treatments such as vaccination with amyloid antigens and antibodies in PD, Alzheimer's disease and potentially other neurodegeneration ailments. METHODOLOGY/PRINCIPAL FINDINGS: Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies--α-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance. We found significantly higher antibody levels towards monomeric α-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression (P<0.0001. This indicates potential protective role of autoimmunity in maintaining the body homeostasis and clearing protein species whose disbalance may lead to amyloid assembly. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards α-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression (P<0.0001. Pooled IgGs from PD patients and controls interacted also with the amyloid fibrils of Aβ (1-40 and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to the generic amyloid conformational epitope, displaying higher specificity towards human amyloid species associated with neurodegeneration. CONCLUSIONS/SIGNIFICANCE: Our findings may suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major

  1. Nutrient deprivation induces α-synuclein aggregation through endoplasmic reticulum stress response and SREBP2 pathway

    OpenAIRE

    Jiang, Peizhou; Gan, Ming; Lin, Wen-Lang; Yen, Shu-Hui C.

    2014-01-01

    Abnormal accumulation of filamentous α-synuclein (α-syn) in neurons, regarded as Lewy bodies (LBs), are a hallmark of Parkinson disease (PD). Although the exact mechanism(s) underlying LBs formation remains unknown, autophagy and ER stress response have emerged as two important pathways affecting α-syn aggregation. In present study we tested whether cells with the tetracycline-off inducible overexpression of α-syn and accumulating α-syn aggregates can benefit from autophagy activation elicite...

  2. Nutrient deprivation induces α-synuclein aggregation through endoplasmic reticulum stress response and SREBP2 pathway

    OpenAIRE

    Peizhou eJiang; Ming eGan; Wen-Lang eLin; Yen, Shu-Hui C.

    2014-01-01

    Abnormal accumulation of filamentous α-synuclein (α-syn) in neurons, regarded as Lewy bodies(LBs), are a hallmark of Parkinson disease (PD). Although the exact mechanism(s) underlying LBs formation remains unknown, autophagy and ER stress response have emerged as two important pathways affecting α-syn aggregation. In present study we tested whether cells with the tetracycline-off inducible overexpression of α-syn and accumulating α-syn aggregates can benefit from autophagy activation elicited...

  3. Neurotoxicity of the Parkinson Disease-Associated Pesticide Ziram Is Synuclein-Dependent in Zebrafish Embryos

    OpenAIRE

    Lulla, A.; Barnhill, L; Bitan, G.; Ivanova, MI; Nguyen, B; O'Donnell, K.; Stahl, MC; Yamashiro, C.; Klaerner, F-G; Schrader, T; Sagasti, A; Bronstein, JM

    2016-01-01

    Background: Exposure to the commonly used dithiocarbamate (DTC) pesticides is associated with an increased risk of developing Parkinson disease (PD), although the mechanisms by which they exert their toxicity are not completely understood. Objective: We studied the mechanisms of ziram’s (a DTC fungicide) neurotoxicity in vivo. Methods: Zebrafish (ZF) embryos were utilized to determine ziram’s effects on behavior, neuronal toxicity, and the role of synuclein in its toxicity. Results: Nanomolar...

  4. α-Synuclein-induced Aggregation of Cytoplasmic Vesicles in Saccharomyces cerevisiae

    OpenAIRE

    Soper, James H.; Roy, Subhojit; Stieber, Anna; Lee, Eliza; Wilson, Robert B.; Trojanowski, John Q.; Burd, Christopher G.; Lee, Virginia M.-Y.

    2008-01-01

    Aggregated α-synuclein (α-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, α-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human α-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of ye...

  5. Prion-like transmission of α-synuclein pathology in the context of an NFL null background.

    Science.gov (United States)

    Rutherford, Nicola J; Brooks, Mieu; Riffe, Cara J; Gorion, Kimberly-Marie M; Howard, Jasie K; Dhillon, Jess-Karan S; Giasson, Benoit I

    2017-09-28

    Neurofilaments are a major component of the axonal cytoskeleton in neurons and have been implicated in a number of neurodegenerative diseases due to their presence within characteristic pathological inclusions. Their contributions to these diseases are not yet fully understood, but previous studies investigated the effects of ablating the obligate subunit of neurofilaments, low molecular mass neurofilament subunit (NFL), on disease phenotypes in transgenic mouse models of Alzheimer's disease and tauopathy. Here, we tested the effects of ablating NFL in α-synuclein M83 transgenic mice expressing the human pathogenic A53T mutation, by breeding them onto an NFL null background. The induction and spread of α-synuclein inclusion pathology was triggered by the injection of preformed α-synuclein fibrils into the gastrocnemius muscle or hippocampus in M83 versus M83/NFL null mice. We observed no difference in the post-injection time to motor-impairment and paralysis endpoint or amount and distribution of α-synuclein inclusion pathology in the muscle injected M83 and M83/NFL null mice. Hippocampal injected M83/NFL null mice displayed subtle region-specific differences in the amount of α-synuclein inclusions however, pathology was observed in the same regions as the M83 mice. Overall, we observed only minor differences in the induction and transmission of α-synuclein pathology in these induced models of synucleinopathy in the presence or absence of NFL. This suggests that NFL and neurofilaments do not play a major role in influencing the induction and transmission of α-synuclein aggregation. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Transient β-hairpin formation in α-synuclein monomer revealed by coarse-grained molecular dynamics simulation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hang; Ma, Wen [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Han, Wei [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Schulten, Klaus, E-mail: kschulte@ks.uiuc.edu [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States)

    2015-12-28

    Parkinson’s disease, originating from the intrinsically disordered peptide α-synuclein, is a common neurodegenerative disorder that affects more than 5% of the population above age 85. It remains unclear how α-synuclein monomers undergo conformational changes leading to aggregation and formation of fibrils characteristic for the disease. In the present study, we perform molecular dynamics simulations (over 180 μs in aggregated time) using a hybrid-resolution model, Proteins with Atomic details in Coarse-grained Environment (PACE), to characterize in atomic detail structural ensembles of wild type and mutant monomeric α-synuclein in aqueous solution. The simulations reproduce structural properties of α-synuclein characterized in experiments, such as secondary structure content, long-range contacts, chemical shifts, and {sup 3}J(H{sub N}H{sub C{sub α}})-coupling constants. Most notably, the simulations reveal that a short fragment encompassing region 38-53, adjacent to the non-amyloid-β component region, exhibits a high probability of forming a β-hairpin; this fragment, when isolated from the remainder of α-synuclein, fluctuates frequently into its β-hairpin conformation. Two disease-prone mutations, namely, A30P and A53T, significantly accelerate the formation of a β-hairpin in the stated fragment. We conclude that the formation of a β-hairpin in region 38-53 is a key event during α-synuclein aggregation. We predict further that the G47V mutation impedes the formation of a turn in the β-hairpin and slows down β-hairpin formation, thereby retarding α-synuclein aggregation.

  7. Decreasing a-synuclein aggregation by methanolic extract of Centella asiatica in zebrafish Parkinson’s model

    Institute of Scientific and Technical Information of China (English)

    Husnul; Khotimah; Mulyohadi; Ali; Sutiman; Bambang; Sumitro; Mochamad; Aris; Widodo

    2015-01-01

    Objective: To observe the effects of Centella asiatica(C. asiatica) methanolic extract on a-synuclein aggregation and its expression in rotenone-exposed zebra fish.Methods: Zebra fish(Danio rerio) were exposed to 5 m g/L rotenone for 28 days and coincubated with 2.5, 5.0 and 10.0 m g/mL of C. asiatica methanolic extract. The medium was changed every 48 h for maintain the concentration of rotenone and extract. After 28 days zebra fish were sacrificed on the ice block and protein was isolated from zebra fish brain for ELISA of dopamine and Western blotting of a-synuclein. Immunohistochemistry was conducted to observe the a-synuclein expressions from histopathological preparation of zebra fish brain. The head were soaked in 10% formaline for less than 24 h and embedded onto paraffin block, then sliced for immunohistochemistry using anti a-synuclein antibody. We also measured zebra fish motility for 5 min in each week.Results: C. asiatica has important bioactive compounds such as asiaticoside that has antiin flammatory and antioxidant properties. It may inhibit cascade reaction due to oxidative stress induced by rotenone. Decreasing reactive oxygen species proposed probability of radical attack to a-synuclein protein that caused aggregation and increase of its expression.The motility of zebra fish was also maintained in C. asiatica groups due to the increasing dopamine level in rotenone-induced zebra fish. High level of reactive oxygen species inactivated enzyme for dopamine synthesis such as tyrosine hydroxylase, and oxidized dopamine itself. Oxidized dopamine increased a-synuclein aggregation. Thus, the dopamine level decreased in rotenone-induced zebra fish, but C. asiatica increased dopamine level.Conclusions: C. asiatica has a potential to be developed as an anti-Parkinson’s disease treatment due to its capability for minimized the sign of Parkinson’s such as a-synuclein aggregation and expression, increasing motility and dopamine as well.

  8. Decreasingα-synuclein aggregation by methanolic extract of Centella asiatica in zebraifsh Parkinson’s model

    Institute of Scientific and Technical Information of China (English)

    Husnul Khotimah; Mulyohadi Ali; Sutiman Bambang Sumitro; Mochamad Aris Widodo

    2015-01-01

    Objective:To observe the effects of Centella asiatica (C. asiatica) methanolic extract onα-synuclein aggregation and its expression in rotenone-exposed zebrafish. Methods: Zebrafish (Danio rerio) were exposed to 5 µg/L rotenone for 28 days and co-incubated with 2.5, 5.0 and 10.0 µg/mL of C. asiatica methanolic extract. The medium was changed every 48 h for maintain the concentration of rotenone and extract. After 28 days zebrafish were sacrificed on the ice block and protein was isolated from zebrafish brain for ELISA of dopamine and Western blotting of α-synuclein. Immunohistochemistry was conducted to observe the α-synuclein expressions from histopathological preparation of zebrafish brain. The head were soaked in 10%formaline for less than 24 h and embedded onto paraffin block, then sliced for immunohistochemistry using antiα-synuclein antibody. We also measured zebrafish motility for 5 min in each week. Results:C. asiatica has important bioactive compounds such as asiaticoside that has anti-inflammatory and antioxidant properties. It may inhibit cascade reaction due to oxidative stress induced by rotenone. Decreasing reactive oxygen species proposed probability of radical attack toα-synuclein protein that caused aggregation and increase of its expression. The motility of zebrafish was also maintained in C. asiatica groups due to the increasing dopamine level in rotenone-induced zebrafish. High level of reactive oxygen species inactivated enzyme for dopamine synthesis such as tyrosine hydroxylase, and oxidized dopamine itself. Oxidized dopamine increasedα-synuclein aggregation. Thus, the dopamine level decreased in rotenone-induced zebrafish, but C. asiatica increased dopamine level. Conclusions: C. asiatica has a potential to be developed as an anti-Parkinson's disease treatment due to its capability for minimized the sign of Parkinson’s such asα-synuclein aggregation and expression, increasing motility and dopamine as well.

  9. Accumulation of α-synuclein in the bowel of patients in the pre-clinical phase of Parkinson's disease.

    Science.gov (United States)

    Hilton, David; Stephens, Madeleine; Kirk, Leanne; Edwards, Philip; Potter, Ross; Zajicek, John; Broughton, Ellie; Hagan, Hannah; Carroll, Camille

    2014-02-01

    Parkinson's disease primarily affects the central nervous system, but autopsy and small patient studies have revealed autonomic nervous system pathology in most cases. We looked for α-synuclein pathology in routinely acquired biopsies from patients and matched controls. Immunocytochemistry was performed and assessed blind to the clinical diagnoses. One hundred and seventeen gastrointestinal tissue samples from 62 patients, and 161 samples from 161 controls, were examined. Twelve biopsies from seven patients showed accumulation of α-synuclein within mucosal and submucosal nerve fibres, and ganglia, which was more extensive with an antibody to phosphorylated, than with an antibody to non-phosphorylated, α-synuclein. These included gastric, duodenal and colonic biopsies, and were taken up to 8 years prior to the onset of motor symptoms. All patients with positive biopsies had early autonomic symptoms and all controls were negative. This large scale study demonstrates that accumulation of α-synuclein in the gastrointestinal tract is a highly specific finding that could be used to confirm a clinical diagnosis of Parkinson's disease. We have shown that α-synuclein accumulation occurs prior to the onset of motor symptoms in the upper, as well as the lower gastrointestinal tract, remains present in serial biopsies until the onset of motor symptoms and is predominantly composed of phosphorylated α-synuclein. Accumulation of α-synuclein in the bowel therefore offers an accessible biomarker which allows further study of the early stages of the disease and could be of value in the assessment of disease modifying treatments.

  10. Identification and quantification of a novel nitrate-reducing community in sediments of Suquia River basin along a nitrate gradient

    Energy Technology Data Exchange (ETDEWEB)

    Reyna, Luciana; Wunderlin, Daniel Alberto [Universidad Nacional de Cordoba-CONICET, Facultad de Ciencias Quimicas, Departamento de Bioquimica Clinica-CIBICI, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Genti-Raimondi, Susana, E-mail: sgenti@fcq.unc.edu.a [Universidad Nacional de Cordoba-CONICET, Facultad de Ciencias Quimicas, Departamento de Bioquimica Clinica-CIBICI, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina)

    2010-05-15

    We evaluated the molecular diversity of narG gene from Suquia River sediments to assess the impact of the nitrate concentration and water quality on the composition and structure of the nitrate-reducing bacterial community. To this aim, a library of one of the six monitoring stations corresponding to the highest nitrate concentration was constructed and 118 narG clones were screened. Nucleotide sequences were associated to narG gene from alpha-, beta-, delta-, gammaproteobacteria and Thermus thermophilus. Remarkably, 18% of clones contained narG genes with less than 69% similarity to narG sequences available in databases. Thus, indicating the presence of nitrate-reducing bacteria with novel narG genes, which were quantified by real-time PCR. Results show a variable number of narG copies, ranging from less than 1.0 x 10{sup 2} to 5.0 x 10{sup 4} copies per ng of DNA, which were associated with a decreased water quality index monitored along the basin at different times. - A novel narG community present in Suquia River sediments was quantified; values were in line with the water quality index.

  11. Enhanced Autophagy from Chronic Toxicity of Iron and Mutant A53T α-Synuclein

    Science.gov (United States)

    Chew, Katherine C. M.; Ang, Eng-Tat; Tai, Yee Kit; Tsang, Fai; Lo, Shun Qiang; Ong, Elijah; Ong, Wei-Yi; Shen, Han-Ming; Lim, Kah-Leong; Dawson, Valina L.; Dawson, Ted M.; Soong, Tuck Wah

    2011-01-01

    Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe2+ uptake, which concomitantly promotes cell death. This Fe2+-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe2+-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe2+ overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe2+-mediated cell death. PMID:21795716

  12. Proteolytic clearance of extracellular α-synuclein as a new therapeutic approach against Parkinson disease.

    Science.gov (United States)

    Park, Sang Myun; Kim, Kwang Soo

    2013-01-01

    Many neurodegenerative diseases such as Alzheimer disease and Parkinson disease show similar characteristics. They typically show deposits of protein aggregates, the formation of which is considered important in their pathogenesis. Recently, aggregation-prone proteins have been shown to spread between cells and so may contribute to the pathogenesis of diseases like prion disease. Such a pathogenesis pathway is possibly common to many neurodegenerative diseases. If confirmed, it could allow the development of therapeutic interventions against many such diseases. In Parkinson disease, α-synuclein, a major component of cytosolic protein inclusions named Lewy body, has been shown to be released and taken up by cells, which may facilitate its progressive pathological spreading between cells. Accordingly, inhibition of spreading by targeting extracellular α-synuclein may represent a new therapy against Parkinson disease. Research into the intercellular spreading of extracellular protein aggregations of α-synuclein and its clearance pathway are reviewed here with a focus on the proteolytic clearance pathway as a therapeutic target for the treatment of Parkinson disease. Considering the similar characteristics of aggregation-prone proteins, these clearance systems might allow treatment of other neurodegenerative diseases beyond Parkinson disease.

  13. Myelination transition zone astrocytes are constitutively phagocytic and have synuclein dependent reactivity in glaucoma.

    Science.gov (United States)

    Nguyen, Judy V; Soto, Ileana; Kim, Keun-Young; Bushong, Eric A; Oglesby, Ericka; Valiente-Soriano, Francisco J; Yang, Zhiyong; Davis, Chung-ha O; Bedont, Joseph L; Son, Janice L; Wei, John O; Buchman, Vladimir L; Zack, Donald J; Vidal-Sanz, Manuel; Ellisman, Mark H; Marsh-Armstrong, Nicholas

    2011-01-18

    Optic nerve head (ONH) astrocytes have been proposed to play both protective and deleterious roles in glaucoma. We now show that, within the postlaminar ONH myelination transition zone (MTZ), there are astrocytes that normally express Mac-2 (also known as Lgals3 or galectin-3), a gene typically expressed only in phagocytic cells. Surprisingly, even in healthy mice, MTZ and other ONH astrocytes constitutive internalize large axonal evulsions that contain whole organelles. In mouse glaucoma models, MTZ astrocytes further up-regulate Mac-2 expression. During glaucomatous degeneration, there are dystrophic processes in the retina and optic nerve, including the MTZ, which contain protease resistant γ-synuclein. The increased Mac-2 expression by MTZ astrocytes during glaucoma likely depends on this γ-synuclein, as mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ after elevation of intraocular pressure. These results suggest the possibility that a newly discovered normal degradative pathway for axons might contribute to glaucomatous neurodegeneration.

  14. Differences in the binding of copper(I) to α- and β-synuclein.

    Science.gov (United States)

    De Ricco, Riccardo; Valensin, Daniela; Dell'Acqua, Simone; Casella, Luigi; Gaggelli, Elena; Valensin, Gianni; Bubacco, Luigi; Mangani, Stefano

    2015-01-05

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of abnormal α-synuclein (αS) deposits in the brain. Alterations in homeostasis and metal-induced oxidative stress may play a crucial role in the progression of αS amyloid assembly and pathogenesis of PD. Contrary to αS, β-synuclein (βS) is not involved in the PD etiology. However, it has been suggested that the βS/αS ratio is altered in PD, indicating that a correct balance of these two proteins is implicated in the inhibition of αS aggregation. αS and βS share similar abilities to coordinate Cu(II). In this study, we investigated and compared the interaction of Cu(I) with the N-terminal portion of βS and αS by means of NMR, circular dichroism, and X-ray absorption spectroscopies. Our data show the importance of M10K mutation, which induces different Cu(I) chemical environments. Coordination modes 3S1O and 2S2O were identified for βS and αS, respectively. These new insights into the bioinorganic chemistry of copper and synuclein proteins are a basis to understand the molecular mechanism by which βS might inhibit αS aggregation.

  15. Bilateral upregulation of α-synuclein expression in the mouse substantia nigra by intracranial rotenone treatment.

    Science.gov (United States)

    Carriere, Candace H; Kang, Na Hyea; Niles, Lennard P

    2017-02-01

    The pesticide rotenone has been shown to cause systemic inhibition of mitochondrial complex I activity, with consequent degeneration of dopamine neurons along the nigrostriatal pathway, as observed in Parkinson's disease (PD). Recently, intracranial infusion of rotenone was found to increase the protein levels of the Lewy body constituents, α-synuclein and small ubiquitin-related modifier-1(SUMO-1), in the lesioned hemisphere of the mouse brain. These findings are supportive of a mouse model of PD, but information about the dopamine-synthesizing enzyme, tyrosine hydroxylase (TH), an essential marker of dopaminergic status, was not reported. Clarification of this issue is important because an intracranial rotenone mouse model of Parkinson's disease has not been established. Towards this end, the present study examined the effects of intracranial rotenone treatment on TH and α-synuclein immunohistochemistry in addition to forelimb motor function. Mice were unilaterally infused with either vehicle or rotenone (2μg/site) in both the medial forebrain bundle and the substantia nigra. The forelimb asymmetry (cylinder) test indicated a significant decrease in use of the contralateral forelimb in lesioned animals as compared to the sham group. Densitometric analysis revealed a significant depletion of TH immunofluorescence within the ipsilateral striatum and substantia nigra of lesioned animals. Moreover, a significant bilateral increase in α-synuclein immunofluorescence was found in the substantia nigra of lesioned mice, as compared to control animals. These findings indicate that this intracranial rotenone mouse model will be useful for studies of neurodegenerative disorders such as PD.

  16. Synphilin-1-binding protein NUB1 is colocalized with nonfibrillar, proteinase K-resistant α-synuclein in presynapses in Lewy body disease.

    Science.gov (United States)

    Tanji, Kunikazu; Mori, Fumiaki; Kito, Katsumi; Kakita, Akiyoshi; Mimura, Junsei; Itoh, Ken; Takahashi, Hitoshi; Kamitani, Tetsu; Wakabayashi, Koichi

    2011-10-01

    α-Synuclein is a major component of Lewy bodies in Parkinson disease (PD) and dementia with Lewy bodies (DLB). We recently showed that abnormal α-synuclein with resistance to proteinase K (PK) is deposited at presynapses of distinct brain anatomic regions from the early stages of PD and DLB. NUB1, a synphilin-1-binding protein, also accumulates in Lewy bodies, but it is not known whether abnormal α-synuclein is associated with NUB1. Here, we demonstrate that, in the brain of patients with PD and DLB, NUB1 accumulates in the presynapses in the hippocampus, cerebral neocortex, and substantia nigra in which PK-resistant α-synuclein is deposited. Endogenous NUB1 also accumulated with PK-resistant α-synuclein in the presynapses of transgenic mice that express human α-synuclein with an A53T mutation. Immunoelectron microscopy showed that NUB1 is localized to presynaptic nerve terminals where no abnormal filaments are seen. Biochemical analyses showed that NUB1 coexists with abnormal α-synuclein in the brain of DLB patients. These findings suggest that NUB1 along with abnormal α-synuclein is involved in the pathogenesis of Lewy body disease.

  17. Tubulin Polymerization Promoting Protein (TPPP/p25α) promotes unconventional secretion of α-synuclein through exophagy by impairing autophagosome-lysosome fusion

    DEFF Research Database (Denmark)

    Ejlerskov, Patrick; Rasmussen, Izabela Zorawska; Tolstrup Nielsen, Troels;

    2013-01-01

    , and that both lysosomal fusion block and secretion of α-synuclein could be replicated by knockdown of the p25α target, HDAC6, the predominant cytosolic deacetylase in neurons. Our data indicate that unconventional secretion of α-synuclein can be mediated through exophagy and that factors, which increase...

  18. Pressure effects on α-synuclein amyloid fibrils: An experimental investigation on their dissociation and reversible nature.

    Science.gov (United States)

    Piccirilli, Federica; Plotegher, Nicoletta; Spinozzi, Francesco; Bubacco, Luigi; Mariani, Paolo; Beltramini, Mariano; Tessari, Isabella; Militello, Valeria; Perucchi, Andrea; Amenitsch, Heinz; Baldassarri, Enrico; Steinhart, Milos; Lupi, Stefano; Ortore, Maria Grazia

    2017-08-01

    α-synuclein amyloid fibrils are found in surviving neurons of Parkinson's disease affected patients, but the role they play in the disease development is still under debate. A growing number of evidences points to soluble oligomers as the major cytotoxic species, while insoluble fibrillar aggregates could even play a protection role. In this work, we investigate α-synuclein fibrils dissociation induced at high pressure by means of Small Angle X-ray Scattering and Fourier Transform Infrared Spectroscopy. Fibrils were produced from wild type α-synuclein and two familial mutants, A30P and A53T. Our results enlighten the different reversible nature of α-synuclein fibrils fragmentation at high pressure and suggest water excluded volumes presence in the fibrils core. Wild type and A30P species stabilized at high pressure are highly amyloidogenic and quickly re-associate into fibrils upon decompression, while A53T species shows a partial reversibility of the process likely due to the presence of an intermediate oligomeric state stabilized at high pressure. The amyloid fibrils dissociation process is here suggested to be associated to a negative activation volume, supporting the notion that α-synuclein fibrils are in a high-volume and high-compressibility state and hinting at the presence of a hydration-mediated activated state from which dissociation occurs. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Transmission of prion strains in a transgenic mouse model overexpressing human A53T mutated α-synuclein.

    Science.gov (United States)

    Mougenot, Anne-Laure J; Bencsik, Anna; Nicot, Simon; Vulin, Johann; Morignat, Eric; Verchère, Jérémy; Bétemps, Dominique; Lakhdar, Latefa; Legastelois, Stéphane; Baron, Thierry G

    2011-05-01

    There is a growing interest in the potential roles of misfolded protein interactions in neurodegeneration. To investigate this issue, we inoculated 3 prion strains intracerebrally into transgenic (TgM83) mice that overexpress human A53T α-synuclein. In comparison to nontransgenic controls, there was a striking decrease in the incubation periods of scrapie, classic and H-type bovine spongiform encephalopathies(C-BSE and H-BSE), with conservation of the histopathologic and biochemical features characterizing these 3 prion strains. TgM83 mice died of scrapie or C-BSE prion diseases before accumulating the insoluble and phosphorylated forms of α-synuclein specific to late stages of synucleinopathy. In contrast, the median incubation time for TgM83 mice inoculated with H-BSE was comparable to that observed when these mice were uninfected, thereby allowing the development of molecular alterations of α-synuclein. The last 4 mice of this cohort exhibited early accumulations of H-BSE prion protein along with α-synuclein pathology. The results indicate that a prion disease was triggered concomitantly with an overt synucleinopathy in some transgenic mice overexpressing human A53T α-synuclein after intracerebral inoculation with an H-BSE prion strain.

  20. The chaperonin CCT inhibits assembly of α-synuclein amyloid fibrils by a specific, conformation-dependent interaction

    Science.gov (United States)

    Sot, Begoña; Rubio-Muñoz, Alejandra; Leal-Quintero, Ahudrey; Martínez-Sabando, Javier; Marcilla, Miguel; Roodveldt, Cintia; Valpuesta, José M.

    2017-01-01

    The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington’s disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson’s disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism. PMID:28102321

  1. Short-term effects of a high nitrate diet on nitrate metabolism in healthy individuals.

    Science.gov (United States)

    Bondonno, Catherine P; Liu, Alex H; Croft, Kevin D; Ward, Natalie C; Puddey, Ian B; Woodman, Richard J; Hodgson, Jonathan M

    2015-03-12

    Dietary nitrate, through the enterosalivary nitrate-nitrite-NO pathway, can improve blood pressure and arterial stiffness. How long systemic nitrate and nitrite remain elevated following cessation of high nitrate intake is unknown. In 19 healthy men and women, the time for salivary and plasma nitrate and nitrite to return to baseline after 7 days increased nitrate intake from green leafy vegetables was determined. Salivary and plasma nitrate and nitrite was measured at baseline [D0], end of high nitrate diet [D7], day 9 [+2D], day 14 [+7D] and day 21 [+14D]. Urinary nitrite and nitrate was assessed at D7 and +14D. Increased dietary nitrate for 7 days resulted in a more than fourfold increase in saliva and plasma nitrate and nitrite (p nitrate had returned to baseline while saliva nitrate and nitrite were more than 1.5 times higher than at baseline levels. By [+7D] all metabolites had returned to baseline levels. The pattern of response was similar between men and women. Urinary nitrate and nitrate was sevenfold higher at D7 compared to +14D. These results suggest that daily ingestion of nitrate may be required to maintain the physiological changes associated with high nitrate intake.

  2. The role of quercetin on the survival of neuron-like PC12 cells and the expression of α-synuclein

    Directory of Open Access Journals (Sweden)

    Tae-Beom Ahn

    2015-01-01

    Full Text Available Both genetic and environmental factors are important in the pathogenesis of Parkinson′s disease. As α-synuclein is a major constituent of Lewy bodies, a pathologic hallmark of Parkinson′s disease, genetic aspects of α-synuclein is widely studied. However, the influence of dietary factors such as quercetin on α-synuclein was rarely studied. Herein we aimed to study the neuroprotective role of quercetin against various toxins affecting apoptosis, autophagy and aggresome, and the role of quercetin on α-synuclein expression. PC12 cells were pre-treated with quercetin (100, 500, 1,000 μM and then together with various drugs such as 1-methyl-4-phenylpyridinium (MPP + ; a free radical generator, 6-hydroxydopamine (6-OHDA; a free radical generator, ammonium chloride (an autophagy inhibitor, and nocodazole (an aggresome inhibitor. Cell viability was determined using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertazolium bromide (MTT assay. Apoptosis was detected by annexin V-fluorescein isothiocyanate and propidium iodide through the use of fluorescence activated cell sorter. α-Synuclein expression was detected by western blot assay and immunohistochemistry. The role of α-synuclein was further studied by knocking out α-synuclein using RNA interference. Cell viability increased at lower concentrations (100 and 500 μM of quercetin but decreased at higher concentration (1,000 μM. Quercetin exerted neuroprotective effect against MPP + , ammonium chloride and nocodazole at 100 μM. MPP + induced apoptosis was decreased by 100 μM quercetin. Quercetin treatment increased α-synuclein expression. However, knocking out α-synuclein exerted no significant effect on cell survival. In conclusion, quercetin is neuroprotective against toxic agents via affecting various mechanisms such as apoptosis, autophagy and aggresome. Because α-synuclein expression is increased by quercetin, the role of quercetin as an environmental factor in Parkinson′s disease

  3. The role of quercetin on the survival of neuron-like PC12 cells and the expression ofα-synuclein

    Institute of Scientific and Technical Information of China (English)

    Tae-Beom Ahn; Beom S Jeon

    2015-01-01

    Both genetic and environmental factors are important in the pathogenesis of Parkinson’s disease. Asα-synuclein is a major constituent of Lewy bodies, a pathologic hallmark of Parkinson’s dis-ease, genetic aspects ofα-synuclein is widely studied. However, the inlfuence of dietary factors such as quercetin onα-synuclein was rarely studied. Herein we aimed to study the neuropro-tective role of quercetin against various toxins affecting apoptosis, autophagy and aggresome, and the role of quercetin onα-synuclein expression. PC12 cells were pre-treated with quercetin (100, 500, 1,000 μM) and then together with various drugs such as 1-methyl-4-phenylpyridin-ium (MPP+; a free radical generator), 6-hydroxydopamine (6-OHDA; a free radical generator), ammonium chloride (an autophagy inhibitor), and nocodazole (an aggresome inhibitor). Cell viability was determined using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertazolium bromide (MTT) assay. Apoptosis was detected by annexin V-lfuorescein isothiocyanate and propidium iodide through the use of lfuorescence activated cell sorter.α-Synuclein expression was detected by western blot assay and immunohistochemistry. The role of α-synuclein was further studied by knocking outα-synuclein using RNA interference. Cell viability increased at lower concen-trations (100 and 500 μM) of quercetin but decreased at higher concentration (1,000 μM). Quercetin exerted neuroprotective effect against MPP+, ammonium chloride and nocodazole at 100 μM. MPP+ induced apoptosis was decreased by 100 μM quercetin. Quercetin treatment in-creasedα-synuclein expression. However, knocking outα-synuclein exerted no signiifcant effect on cell survival. In conclusion, quercetin is neuroprotective against toxic agentsvia affecting var-ious mechanisms such as apoptosis, autophagy and aggresome. Becauseα-synuclein expression is increased by quercetin, the role of quercetin as an environmental factor in Parkinson’s disease pathogenesis needs

  4. Efficiency of nitrate uptake in spinach : impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

    NARCIS (Netherlands)

    Ter Steege, MW; Stulen, [No Value; Wiersema, PK; Posthumus, F; Vaalburg, W

    1999-01-01

    Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with N-13- and N-15-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration. Plan

  5. Metal nitrate conversion method, patent application

    NARCIS (Netherlands)

    2008-01-01

    A method for converting a supported metal nitrate into the corresponding supported metal comprises heating the metal nitrate to effect its decomposition under a gas mixture that contains nitric oxide and has an oxygen content of

  6. 76 FR 62311 - Ammonium Nitrate Security Program

    Science.gov (United States)

    2011-10-07

    ... to best notify agents (AN Agents) when ammonium nitrate purchasers (AN Purchasers) submit those AN... directly to ammonium nitrate sellers (AN Sellers) when it is not possible for an AN Seller to verify the...

  7. Progressive neurodegenerative and behavioural changes induced by AAV-mediated overexpression of α-synuclein in midbrain dopamine neurons

    DEFF Research Database (Denmark)

    Decressac, M; Mattsson, Bente; Lundblad, M

    2012-01-01

    Parkinson's disease (PD) is characterised by the progressive loss of nigral dopamine neurons and the presence of synucleinopathy. Overexpression of α-synuclein in vivo using viral vectors has opened interesting possibilities to model PD-like pathology in rodents. However, the attempts made so far...... have failed to show a consistent behavioural phenotype and pronounced dopamine neurodegeneration. Using a more efficient adeno-associated viral (AAV) vector construct, which includes a WPRE enhancer element and uses the neuron-specific synapsin-1 promoter to drive the expression of human wild-type α......-synuclein, we have now been able to achieve increased levels of α-synuclein in the transduced midbrain dopamine neurons sufficient to induce profound deficits in motor function, accompanied by reduced expression of proteins involved in dopamine neurotransmission and a time-dependent loss of nigral dopamine...

  8. End-to-end Structural Restriction of α-Synuclein and Its Influence on Amyloid Fibril Formation

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Chul Suk; Park, Jae Hyung; Choe, Young Jun; Paik, Seung R. [Seoul National University, Seoul (Korea, Republic of)

    2014-09-15

    Relationship between molecular freedom of amyloidogenic protein and its self-assembly into amyloid fibrils has been evaluated with α-synuclein, an intrinsically unfolded protein related to Parkinson's disease, by restricting its structural plasticity through an end-to-end disulfide bond formation between two newly introduced cysteine residues on the N- and C-termini. Although the resulting circular form of α-synuclein exhibited an impaired fibrillation propensity, the restriction did not completely block the protein's interactive core since co-incubation with wild-type α-synuclein dramatically facilitated the fibrillation by producing distinctive forms of amyloid fibrils. The suppressed fibrillation propensity was instantly restored as the structural restriction was unleashed with β-mercaptoethanol. Conformational flexibility of the accreting amyloidogenic protein to pre-existing seeds has been demonstrated to be critical for fibrillar extension process by exerting structural adjustment to a complementary structure for the assembly.

  9. Nitrate Removal from Ground Water: A Review

    OpenAIRE

    Archna *; Surinder K. Sharma; Ranbir Chander Sobti

    2012-01-01

    Nitrate contamination of ground water resources has increased in Asia, Europe, United States, and various other parts of the world. This trend has raised concern as nitrates cause methemoglobinemia and cancer. Several treatment processes can remove nitrates from water with varying degrees of efficiency, cost, and ease of operation. Available technical data, experience, and economics indicate that biological denitrification is more acceptable for nitrate removal than reverse osmosis and ion ex...

  10. NITRATE + NITRITE CONTENT (CONCENTRATION), PHOSPHATE, NITRITE, SILICATE and other profile and discrete sample data collected in the Gulf of Alaska on the ALPHA HELIX and WECOMA cruises HX203, HX205 and others as part of the NEP project from 1998-03-08 to 2004-07-05 (NODC Accession 0115260)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — NODC Accession 0115260 includes profile, discrete sample and chemical data collected aboard the ALPHA HELIX and WECOMA during cruises HX203, HX205, HX208, HX211,...

  11. 49 CFR 176.410 - Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures. 176.410 Section 176.410 Transportation Other Regulations Relating to... nitrate and ammonium nitrate mixtures. (a) This section prescribes requirements to be observed...

  12. Nitrate tolerance impairs nitric oxide-mediated vasodilation in vivo

    DEFF Research Database (Denmark)

    Laursen, Jørn Bech; Boesgaard, Søren; Poulsen, Henrik E.;

    1996-01-01

    Nitrates, Nitrate tolerence, Nitric oxide, acetylcholine, N-acetylcholine, N-acetylcysteine, L-NAME, Rat, Anesthetized......Nitrates, Nitrate tolerence, Nitric oxide, acetylcholine, N-acetylcholine, N-acetylcysteine, L-NAME, Rat, Anesthetized...

  13. 76 FR 11273 - Ammonium Nitrate From Russia

    Science.gov (United States)

    2011-03-01

    ... COMMISSION Ammonium Nitrate From Russia AGENCY: United States International Trade Commission. ACTION: Institution of a five-year review concerning the suspended investigation on ammonium nitrate from Russia... investigation on ammonium nitrate from Russia would be likely to lead to continuation or recurrence of...

  14. 76 FR 47238 - Ammonium Nitrate From Russia

    Science.gov (United States)

    2011-08-04

    ... COMMISSION Ammonium Nitrate From Russia Determination On the basis of the record \\1\\ developed in the subject... order on ammonium nitrate from Russia would be likely to lead to continuation or recurrence of material... Commission are contained in USITC Publication 4249 (August 2011), entitled Ammonium Nitrate from...

  15. 21 CFR 172.160 - Potassium nitrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium nitrate. 172.160 Section 172.160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Preservatives § 172.160 Potassium nitrate. The food additive potassium nitrate may be safely used as a...

  16. Conditional transgenic mice expressing C-terminally truncated human α-synuclein (αSyn119 exhibit reduced striatal dopamine without loss of nigrostriatal pathway dopaminergic neurons

    Directory of Open Access Journals (Sweden)

    Flint Beal M

    2009-07-01

    Full Text Available Abstract Background Missense mutations and multiplications of the α-synuclein gene cause autosomal dominant familial Parkinson's disease (PD. α-Synuclein protein is also a major component of Lewy bodies, the hallmark pathological inclusions of PD. Therefore, α-synuclein plays an important role in the pathogenesis of familial and sporadic PD. To model α-synuclein-linked disease in vivo, transgenic mouse models have been developed that express wild-type or mutant human α-synuclein from a variety of neuronal-selective heterologous promoter elements. These models exhibit a variety of behavioral and neuropathological features resembling some aspects of PD. However, an important deficiency of these models is the observed lack of robust or progressive nigrostriatal dopaminergic neuronal degeneration that is characteristic of PD. Results We have developed conditional α-synuclein transgenic mice that can express A53T, E46K or C-terminally truncated (1–119 human α-synuclein pathological variants from the endogenous murine ROSA26 promoter in a Cre recombinase-dependent manner. Using these mice, we have evaluated the expression of these α-synuclein variants on the integrity and viability of nigral dopaminergic neurons with age. Expression of A53T α-synuclein or truncated αSyn119 selectively in nigrostriatal pathway dopaminergic neurons for up to 12 months fails to precipitate dopaminergic neuronal loss in these mice. However, αSyn119 expression in nigral dopaminergic neurons for up to 12 months causes a marked reduction in the levels of striatal dopamine and its metabolites together with other subtle neurochemical alterations. Conclusion We have developed and evaluated novel conditional α-synuclein transgenic mice with transgene expression directed selectively to nigrostriatal dopaminergic neurons as a potential new mouse model of PD. Our data support the pathophysiological relevance of C-terminally truncated α-synuclein species in vivo. The

  17. Proteomic identification of age-dependent protein nitration in rat skeletal muscle.

    Science.gov (United States)

    Kanski, Jaroslaw; Alterman, Michail A; Schöneich, Christian

    2003-11-15

    Age-related protein nitration was studied in skeletal muscle of Fisher 344 and Fisher 344/Brown Norway (BN) F1 rats by a proteomic approach. Proteins from young (4 months) and old (24 months) Fisher 344 rats and young (6 months) and old (34 months) Fisher 344/BN F1 animals were separated by 2-D gel electrophoresis. Western blot showed an age-related increase in the nitration of a few specific proteins, which were identified by MALDI-TOF MS and ESI-MS/MS. We identified age-dependent apparent nitration of beta-enolase, alpha-fructose aldolase, and creatine kinase, which perform important functions in muscle energy metabolism, suggesting that the nitration of such key proteins can be, in part, responsible for the decline of muscle motor function of the muscle. Furthermore, we have identified the apparent nitration of succinate dehydrogenase, rab GDP dissociation inhibitor beta (GdI-2), triosephosphate isomerase, troponin I, alpha-crystallin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

  18. Negative feedback loops leading to nitrate homeostasis and oscillatory nitrate assimilation in plants and fungi.

    OpenAIRE

    Huang, Yongshun

    2011-01-01

    Nitrate is an important nutrient for plants and fungi. For plants it has been shown that cytosolic nitrate levels are under homeostatic control. Here we describe two networks that can obtain robust, i.e. perturbation independent, homeostatic behavior in cytosolic nitrate concentration. One of the networks, a member in the family of outflow controllers, is based on a negative feedback loop containing a nitrate-induced activation of a controller molecule which removes nitrate. In plants this co...

  19. A Spirulina-Enhanced Diet Provides Neuroprotection in an α-Synuclein Model of Parkinson's Disease

    Science.gov (United States)

    Pabon, Mibel M.; Jernberg, Jennifer N.; Morganti, Josh; Contreras, Jessika; Hudson, Charles E.; Klein, Ronald L.; Bickford, Paula C.

    2012-01-01

    Inflammation in the brain plays a major role in neurodegenerative diseases. In particular, microglial cell activation is believed to be associated with the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD). An increase in microglia activation has been shown in the substantia nigra pars compacta (SNpc) of PD models when there has been a decrease in tyrosine hydroxylase (TH) positive cells. This may be a sign of neurotoxicity due to prolonged activation of microglia in both early and late stages of disease progression. Natural products, such as spirulina, derived from blue green algae, are believed to help reverse this effect due to its anti-inflammatory/anti-oxidant properties. An adeno-associated virus vector (AAV9) for α-synuclein was injected in the substantia nigra of rats to model Parkinson's disease and to study the effects of spirulina on the inflammatory response. One month prior to surgeries, rats were fed either a diet enhanced with spirulina or a control diet. Immunohistochemistry was analyzed with unbiased stereological methods to quantify lesion size and microglial activation. As hypothesized, spirulina was neuroprotective in this α-synuclein model of PD as more TH+ and NeuN+ cells were observed; spirulina concomitantly decreased the numbers of activated microglial cells as determined by MHCII expression. This decrease in microglia activation may have been due, in part, to the effect of spirulina to increase expression of the fractalkine receptor (CX3CR1) on microglia. With this study we hypothesize that α-synuclein neurotoxicity is mediated, at least in part, via an interaction with microglia. We observed a decrease in activated microglia in the rats that received a spirulina- enhanced diet concomitant to neuroprotection. The increase in CX3CR1 in the groups that received spirulina, suggests a potential mechanism of action. PMID:23028885

  20. A spirulina-enhanced diet provides neuroprotection in an α-synuclein model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Mibel M Pabon

    Full Text Available Inflammation in the brain plays a major role in neurodegenerative diseases. In particular, microglial cell activation is believed to be associated with the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD. An increase in microglia activation has been shown in the substantia nigra pars compacta (SNpc of PD models when there has been a decrease in tyrosine hydroxylase (TH positive cells. This may be a sign of neurotoxicity due to prolonged activation of microglia in both early and late stages of disease progression. Natural products, such as spirulina, derived from blue green algae, are believed to help reverse this effect due to its anti-inflammatory/anti-oxidant properties. An adeno-associated virus vector (AAV9 for α-synuclein was injected in the substantia nigra of rats to model Parkinson's disease and to study the effects of spirulina on the inflammatory response. One month prior to surgeries, rats were fed either a diet enhanced with spirulina or a control diet. Immunohistochemistry was analyzed with unbiased stereological methods to quantify lesion size and microglial activation. As hypothesized, spirulina was neuroprotective in this α-synuclein model of PD as more TH+ and NeuN+ cells were observed; spirulina concomitantly decreased the numbers of activated microglial cells as determined by MHCII expression. This decrease in microglia activation may have been due, in part, to the effect of spirulina to increase expression of the fractalkine receptor (CX3CR1 on microglia. With this study we hypothesize that α-synuclein neurotoxicity is mediated, at least in part, via an interaction with microglia. We observed a decrease in activated microglia in the rats that received a spirulina- enhanced diet concomitant to neuroprotection. The increase in CX3CR1 in the groups that received spirulina, suggests a potential mechanism of action.

  1. A spirulina-enhanced diet provides neuroprotection in an α-synuclein model of Parkinson's disease.

    Science.gov (United States)

    Pabon, Mibel M; Jernberg, Jennifer N; Morganti, Josh; Contreras, Jessika; Hudson, Charles E; Klein, Ronald L; Bickford, Paula C

    2012-01-01

    Inflammation in the brain plays a major role in neurodegenerative diseases. In particular, microglial cell activation is believed to be associated with the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). An increase in microglia activation has been shown in the substantia nigra pars compacta (SNpc) of PD models when there has been a decrease in tyrosine hydroxylase (TH) positive cells. This may be a sign of neurotoxicity due to prolonged activation of microglia in both early and late stages of disease progression. Natural products, such as spirulina, derived from blue green algae, are believed to help reverse this effect due to its anti-inflammatory/anti-oxidant properties. An adeno-associated virus vector (AAV9) for α-synuclein was injected in the substantia nigra of rats to model Parkinson's disease and to study the effects of spirulina on the inflammatory response. One month prior to surgeries, rats were fed either a diet enhanced with spirulina or a control diet. Immunohistochemistry was analyzed with unbiased stereological methods to quantify lesion size and microglial activation. As hypothesized, spirulina was neuroprotective in this α-synuclein model of PD as more TH+ and NeuN+ cells were observed; spirulina concomitantly decreased the numbers of activated microglial cells as determined by MHCII expression. This decrease in microglia activation may have been due, in part, to the effect of spirulina to increase expression of the fractalkine receptor (CX3CR1) on microglia. With this study we hypothesize that α-synuclein neurotoxicity is mediated, at least in part, via an interaction with microglia. We observed a decrease in activated microglia in the rats that received a spirulina- enhanced diet concomitant to neuroprotection. The increase in CX3CR1 in the groups that received spirulina, suggests a potential mechanism of action.

  2. Observations of peroxyacetyl nitrate (PAN) in air in Southern England

    Energy Technology Data Exchange (ETDEWEB)

    Stephens, E.R. (Univ. of California, Riverside); Penkett, S.A.; Sandalls, F.J.; Lovelock, J.E.

    1975-04-01

    It has always seemed inevitable that PAN would be found in any urban or near urban area whenever a sufficiently diligent search with high sensitivity was mounted. Detection in Tokyo, Japan has been reported as well as several locations in the USA. Penkett, Sandalls and Lovelock report a good separation with high sensitivity showing PPN as well as PAN in about the same ratio as seen in California. The correlation with ozone is not always so good. Formation of small concentrations of PAN in natural atmospheres would not be surprising since volatile terpenes such as ..cap alpha..-pinene are very reactive and can form PAN when irradiated with nitrogen oxides. There are also natural sources of nitrogen oxides. Nevertheless, modern autos are such prolific sources of the precursors of PAN and ozone that they should be the prime suspect even in places more remote than Harwell. The chromatogram given by Penkett et al. has a peak labelled methyl nitrate which is not discussed in the text. We have not been able to detect methyl nitrate in Riverside, California. We do see a peak at just half the emergence time of PAN which we identify as tetrachloroethylene (C/sub 2/Cl/sub 4/). This peak shows a very different time variation from PAN or ozone which is not surprising if it is derived from the nearest dry cleaning establishment. If the peak in the Harwell chromatogram is methyl nitrate it would provide an additional tracer for photochemical reaction products. (auth)

  3. Continuous flow nitration in miniaturized devices.

    Science.gov (United States)

    Kulkarni, Amol A

    2014-01-01

    This review highlights the state of the art in the field of continuous flow nitration with miniaturized devices. Although nitration has been one of the oldest and most important unit reactions, the advent of miniaturized devices has paved the way for new opportunities to reconsider the conventional approach for exothermic and selectivity sensitive nitration reactions. Four different approaches to flow nitration with microreactors are presented herein and discussed in view of their advantages, limitations and applicability of the information towards scale-up. Selected recent patents that disclose scale-up methodologies for continuous flow nitration are also briefly reviewed.

  4. Continuous flow nitration in miniaturized devices

    Directory of Open Access Journals (Sweden)

    Amol A. Kulkarni

    2014-02-01

    Full Text Available This review highlights the state of the art in the field of continuous flow nitration with miniaturized devices. Although nitration has been one of the oldest and most important unit reactions, the advent of miniaturized devices has paved the way for new opportunities to reconsider the conventional approach for exothermic and selectivity sensitive nitration reactions. Four different approaches to flow nitration with microreactors are presented herein and discussed in view of their advantages, limitations and applicability of the information towards scale-up. Selected recent patents that disclose scale-up methodologies for continuous flow nitration are also briefly reviewed.

  5. Identification of the Minimal Copper(II)-binding α-Synuclein Sequence

    OpenAIRE

    Jackson, Mark S.; Lee, Jennifer C.

    2009-01-01

    Parkinson’s disease has been long linked to environmental factors, such as transition metals and recently to α-synuclein, a presynaptic protein. Using tryptophan-containing peptides, we identified the minimal Cu(II)-binding sequence to be within the first four residues, MDV(F/W), anchored by the α-amino terminus. In addition, mutant peptide 1–10 (Lys→Arg) verified that neither Lys6 or Lys10 are necessary for Cu(II) binding. Interestingly, Trp4 excited-state decay kinetics measured for peptide...

  6. Copper Coordination to the Membrane Bound Form of α-Synuclein

    OpenAIRE

    Dudzik, Christopher G.; Walter, Eric D; Abrams, Benjamin S.; Jurica, Melissa S.; Millhauser, Glenn L.

    2012-01-01

    Aggregation of the 140 amino acid protein α-synuclein (α-syn) is linked to the development of Parkinson’s disease (PD). α-Syn is a copper binding protein with potential function as a regulator of metal dependent redox activity. Epidemiological studies suggest that human exposure to excess copper increases the incidence of PD. α-Syn exists in both solution and membrane bound forms. Previous work evaluated the Cu2+ uptake for α-syn in solution and identified Met1 Asp2 and His50 as primary contr...

  7. A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity

    Science.gov (United States)

    Suresh, S. N.; Chavalmane, Aravinda K.; DJ, Vidyadhara; Yarreiphang, Haorei; Rai, Shashank; Paul, Abhik; Clement, James P.; Alladi, Phalguni Anand; Manjithaya, Ravi

    2017-01-01

    ABSTRACT Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders. PMID:28350199

  8. Copper(II) Binding to α-Synuclein, the Parkinson’s Protein

    OpenAIRE

    Lee, Jennifer C.; Gray, Harry B.; Winkler, Jay R.

    2008-01-01

    Variations in tryptophan fluorescence intensities confirm that copper(II) interacts with α-synuclein, a protein implicated in Parkinson’s disease. Trp4 fluorescence decay kinetics measured for the F4W protein show that Cu(II) binds tightly (K d ∼ 100 nM) near the N-terminus at pH 7. Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site.

  9. Abnormal colonic motility in mice overexpressing human wild-type α-synuclein

    OpenAIRE

    2008-01-01

    The presynaptic protein α-synuclein (αSyn) has been implicated in both familial and sporadic forms of Parkinson’s disease. We examined whether human αSyn-overexpressing mice under Thy1 promoter (Thy1-αSyn) display alterations of colonic function. Basal fecal output was decreased in Thy1-αSyn mice fed ad libitum. Fasted/refed Thy1-αSyn mice had a slower distal colonic transit than the wild-type mice, as monitored by 2.2-fold increase in time to expel an intracolonic bead and 2.9-fold higher co...

  10. Nitrate metabolism in the gromiid microbial universe

    DEFF Research Database (Denmark)

    Høgslund, Signe; Risgaard-Petersen, Nils; Cedhagen, Tomas

    Eukaryotic nitrate respiration supported by intracellular nitrate storages contributes substantially to the nitrogen cycle. Research focus is currently directed towards two phyla: Foraminifera and diatoms, but the widespread Gromia in the Rhizaria may be another key organism. These giant protists...... enclose and regulate a small biogeochemical universe within their cell. Their transparent proteinaceous cell wall surrounds a complex matrix consisting of sediment, bacteria and nitrate which is concentrated to hundreds of mM in the gromiid cell. The nitrate is respired to dinitrogen, but in contrast...... to the findings of eukaryotic mediated nitrate reduction in some foraminifera and diatoms, nitrate respiration in gromiids seems to be mediated by bacterial endosymbionts. The role of endobionts in nitrate accumulating eukaryotes is of fundamental importance for understanding the evolutionary path...

  11. Phase diagram of ammonium nitrate

    Science.gov (United States)

    Dunuwille, M.; Yoo, C. S.

    2014-05-01

    Ammonium Nitrate (AN) has often subjected to uses in improvised explosive devices, due to its wide availability as a fertilizer and its capability of becoming explosive with slight additions of organic and inorganic compounds. Yet, the origin of enhanced energetic properties of impure AN (or AN mixtures) is neither chemically unique nor well understood -resulting in rather catastrophic disasters in the past1 and thereby a significant burden on safety in using ammonium nitrates even today. To remedy this situation, we have carried out an extensive study to investigate the phase stability of AN at high pressure and temperature, using diamond anvil cells and micro-Raman spectroscopy. The present results confirm the recently proposed phase IV-to-IV' transition above 17 GPa2 and provide new constraints for the melting and phase diagram of AN to 40 GPa and 400 °C.

  12. 49 CFR 176.415 - Permit requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers.

    Science.gov (United States)

    2010-10-01

    ... nitrates, and certain ammonium nitrate fertilizers. 176.415 Section 176.415 Transportation Other... requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers. (a) Except as... Captain of the Port (COTP). (1) Ammonium nitrate UN1942, ammonium nitrate fertilizers containing more...

  13. Mapping the subcellular distribution of α-synuclein in neurons using genetically encoded probes for correlated light and electron microscopy: implications for Parkinson's disease pathogenesis.

    Science.gov (United States)

    Boassa, Daniela; Berlanga, Monica L; Yang, Mary Ann; Terada, Masako; Hu, Junru; Bushong, Eric A; Hwang, Minju; Masliah, Eliezer; George, Julia M; Ellisman, Mark H

    2013-02-06

    Modifications to the gene encoding human α-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of α-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human α-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of α-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of α-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, α-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human α-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that α-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that α-synuclein is involved in processes associated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.

  14. 2-Amino-5-chloropyridinium nitrate

    Directory of Open Access Journals (Sweden)

    Donia Zaouali Zgolli

    2009-11-01

    Full Text Available The title structure, C5H6ClN2+·NO3−, is held together by extensive hydrogen bonding between the NO3− ions and 2-amino-5-chloropyridinium H atoms. The cation–anion N—H...O hydrogen bonds link the ions into a zigzag- chain which develops parallel to the b axis. The structure may be compared with that of the related 2-amino-5-cyanopyridinium nitrate.

  15. Sensitivity of nitrate aerosols to ammonia emissions and to nitrate chemistry: implications for present and future nitrate optical depth

    Directory of Open Access Journals (Sweden)

    F. Paulot

    2015-09-01

    Full Text Available We update and evaluate the treatment of nitrate aerosols in the Geophysical Fluid Dynamics Laboratory (GFDL atmospheric model (AM3. Accounting for the radiative effects of nitrate aerosols generally improves the simulated aerosol optical depth, although nitrate concentrations at the surface are biased high. This bias can be reduced by increasing the deposition of nitrate to account for the near-surface volatilization of ammonium nitrate or by neglecting the heterogeneous production of nitric acid to account for the inhibition of N2O5 reactive uptake at high nitrate concentrations. Globally, uncertainties in these processes can impact the simulated nitrate optical depth by up to 25 %, much more than the impact of uncertainties in the seasonality of ammonia emissions (6 % or in the uptake of nitric acid on dust (13 %. Our best estimate for present-day fine nitrate optical depth at 550 nm is 0.006 (0.005–0.008. We only find a modest increase of nitrate optical depth (2 (−40 % and ammonia (+38 % from 2010 to 2050. Nitrate burden is projected to increase in the tropics and in the free troposphere, but to decrease at the surface in the midlatitudes because of lower nitric acid concentrations. Our results suggest that better constraints on the heterogeneous chemistry of nitric acid on dust, on tropical ammonia emissions, and on the transport of ammonia to the free troposphere are needed to improve projections of aerosol optical depth.

  16. Sensitivity of nitrate aerosols to ammonia emissions and to nitrate chemistry: implications for present and future nitrate optical depth

    Science.gov (United States)

    Paulot, F.; Ginoux, P.; Cooke, W. F.; Donner, L. J.; Fan, S.; Lin, M.-Y.; Mao, J.; Naik, V.; Horowitz, L. W.

    2016-02-01

    We update and evaluate the treatment of nitrate aerosols in the Geophysical Fluid Dynamics Laboratory (GFDL) atmospheric model (AM3). Accounting for the radiative effects of nitrate aerosols generally improves the simulated aerosol optical depth, although nitrate concentrations at the surface are biased high. This bias can be reduced by increasing the deposition of nitrate to account for the near-surface volatilization of ammonium nitrate or by neglecting the heterogeneous production of nitric acid to account for the inhibition of N2O5 reactive uptake at high nitrate concentrations. Globally, uncertainties in these processes can impact the simulated nitrate optical depth by up to 25 %, much more than the impact of uncertainties in the seasonality of ammonia emissions (6 %) or in the uptake of nitric acid on dust (13 %). Our best estimate for fine nitrate optical depth at 550 nm in 2010 is 0.006 (0.005-0.008). In wintertime, nitrate aerosols are simulated to account for over 30 % of the aerosol optical depth over western Europe and North America. Simulated nitrate optical depth increases by less than 30 % (0.0061-0.010) in response to projected changes in anthropogenic emissions from 2010 to 2050 (e.g., -40 % for SO2 and +38 % for ammonia). This increase is primarily driven by greater concentrations of nitrate in the free troposphere, while surface nitrate concentrations decrease in the midlatitudes following lower concentrations of nitric acid. With the projected increase of ammonia emissions, we show that better constraints on the vertical distribution of ammonia (e.g., convective transport and biomass burning injection) and on the sources and sinks of nitric acid (e.g., heterogeneous reaction on dust) are needed to improve estimates of future nitrate optical depth.

  17. Structural Variation of Alpha-synuclein with Temperature by a Coarse-grained Approach with Knowledge-based Interactions (Postprint)

    Science.gov (United States)

    2015-07-01

    Boltzmann constant and the energy (εij), and an attempt to move each residue once defines the unit Monte Carlo step (MCS).35 We monitor a number of local...configuration at the start of the simulation, and the bond length between consecutive nodes varies between 2 and √ 10 in units of lattice constant .36...with the fixed bond length frequently used in lattice simulations.36 Small step (one lattice constant ) moves retain some of the small scale details

  18. The mechanisms of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease

    Science.gov (United States)

    Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanis...

  19. Characterizing the dynamics of alpha-synuclein oligomers using hydrogen/deuterium exchange monitored by mass spectrometry

    DEFF Research Database (Denmark)

    Mysling, Simon; Betzer, Cristine; Jensen, Poul H;

    2013-01-01

    hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS), we have analyzed the structural dynamics of soluble αSN oligomers. The analyzed oligomers were metastable, slowly dissociating to monomers over a period of 21 days, after excess monomer had been removed. The C-terminal region of α......-shielded structure. The protected regions were interspersed by two somewhat more dynamic regions (residues 18-38 and 55-70). In the oligomeric state, the isotopic exchange pattern of the region of residues 35-95 of αSN corresponded well with previous nuclear magnetic resonance and electron paramagnetic resonance...

  20. [Frequency of single nucleotide polymorphisms and alpha-synuclein haplotypes associated with sporadic Parkinson's disease in the Mexican population].

    Science.gov (United States)

    Davila-Ortiz de Montellano, D J; Rodriguez-Violante, M; Fresan, A; Monroy-Jaramillo, N; Yescas-Gomez, P

    2016-10-16

    Introduccion. La enfermedad de Parkinson (EP) es una entidad neurodegenerativa comun de inicio en la etapa adulta. Su incidencia en Mexico se estima en 40-50 casos por 100.000 habitantes/año y constituye la cuarta causa de atencion medica en el Instituto Nacional de Neurologia y Neurocirugia. La proteina alfa-sinucleina, SNCA, es clave en la patologia de la EP y sus polimorfismos se han asociado a un riesgo aumentado de desarrollarla. Objetivo. Evaluar el riesgo que representan los polimorfismos rs2619364, rs2619363, rs2736990, rs7684318, rs17016074, rs356219, rs356220 y rs356203 de SNCA en una muestra de sujetos mexicanos para la EP. Sujetos y metodos. Se evaluaron 171 pacientes con diagnostico de EP y 171 controles pareados por sexo y edad mediante reaccion en cadena de la polimerasa en tiempo real, y se realizo un analisis estadistico para determinar la asociacion de los polimorfismos con la enfermedad. Resultados. Las variantes rs356220, rs356203, rs7684318 y rs2736990 de SNCA estan asociadas a la enfermedad y forman dos haplotipos de riesgo elevado para desarrollar EP esporadica en la poblacion mexicana. Conclusiones. Las variaciones en SNCA son un factor de riesgo para desarrollar EP y pueden ser biomarcadores geneticos especificos para pacientes mestizos mexicanos como herramienta de apoyo diagnostico en la EP esporadica.

  1. 5-HT2A Receptor Binding in the Frontal Cortex of Parkinson's Disease Patients and Alpha-Synuclein Overexpressing Mice

    DEFF Research Database (Denmark)

    Rasmussen, Nadja Bredo; Olesen, Mikkel Vestergaard; Brudek, Tomasz;

    2016-01-01

    The receptor is highly involved in aspects of cognition and executive function and seen to be affected in neurodegenerative diseases like Alzheimer’s disease and related to the disease pathology. Even though Parkinson’s disease (PD) is primarily a motor disorder, reports of impaired executive fun...

  2. Joint analysis of the NACP-REP1 marker within the alpha synuclein gene concludes association with alcohol dependence

    National Research Council Canada - National Science Library

    Bönsch, D; Lederer, T; Reulbach, U; Hothorn, T; Kornhuber, J; Bleich, S

    2005-01-01

    .... This increase is significantly associated with craving, especially obsessive craving. On the basis of these observations, the present study analysed two polymorphic repeats within the NACP gene...

  3. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

    Science.gov (United States)

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E.; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R.; Ross, Ian; Parton, Robert G.; Böcking, Till; Gambin, Yann

    2016-01-01

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils. PMID:27892477

  4. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence.

    Science.gov (United States)

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R; Ross, Ian; Parton, Robert G; Böcking, Till; Gambin, Yann

    2016-11-28

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

  5. Impact of high cholesterol in a Parkinson's disease model: Prevention of lysosomal leakage versus stimulation of α-synuclein aggregation.

    Science.gov (United States)

    Eriksson, Ida; Nath, Sangeeta; Bornefall, Per; Giraldo, Ana Maria Villamil; Öllinger, Karin

    2017-03-01

    Parkinson's disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)), we found that MPP(+)-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP(+)-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinson's disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation. Copyright © 2017 Elsevier GmbH. All rights reserved.

  6. DNA methylation levels of α-synuclein intron 1 in the aging brain.

    Science.gov (United States)

    de Boni, Laura; Riedel, Linda; Schmitt, Ina; Kraus, Theo F J; Kaut, Oliver; Piston, Dominik; Akbarian, Schahram; Wüllner, Ullrich

    2015-12-01

    DNA methylation patterns change with age, and aging itself is a major confounding risk factor for Parkinson's disease (PD). Duplication and triplication, that is, increased expression of the α-synuclein (SNCA) gene, cause familial PD, and demethylation of SNCA intron 1 has been shown to result in increased expression of SNCA. We thus hypothesized that age-related alterations of SNCA methylation might underly the increased susceptibility toward PD in later life. The present study sought to determine (1) whether alterations of SNCA intron 1 methylation occurred during aging, (2) whether the methylation pattern differed between men and women, and (3) whether purified neurons compared with non-neuronal cells exhibited different methylation patterns. The analysis of DNA from brain tissue and fluorescence activated cell sorting-sorted purified neurons of 41 individuals revealed only a minor increase of SNCA intron 1 DNA methylation levels in presumably healthy individuals during aging but no significant difference between men and women. Interestingly enough, methylation of SNCA intron 1 was higher in neurons compared with non-neuronal cells, although non-neuronal cells express lower levels of SNCA. Therefore, the normal pattern of SNCA methylation during aging should not result in increased expression of α-synuclein protein. It is thus likely that additional, yet not identified, mechanisms contribute to the tissue specificity of SNCA expression and the presumed dysregulation in PD.

  7. Neuroinflammation and α-synuclein accumulation in response to glucocerebrosidase deficiency are accompanied by synaptic dysfunction.

    Science.gov (United States)

    Ginns, Edward I; Mak, Sally K-K; Ko, Novie; Karlgren, Juliane; Akbarian, Schahram; Chou, Vivian P; Guo, Yin; Lim, Arlene; Samuelsson, Steven; LaMarca, Mary L; Vazquez-DeRose, Jacqueline; Manning-Boğ, Amy B

    2014-02-01

    Clinical, epidemiological and experimental studies confirm a connection between the common degenerative movement disorder Parkinson's disease (PD) that affects over 1 million individuals, and Gaucher disease, the most prevalent lysosomal storage disorder. Recently, human imaging studies have implicated impaired striatal dopaminergic neurotransmission in early PD pathogenesis in the context of Gaucher disease mutations, but the underlying mechanisms have yet to be characterized. In this report we describe and characterize two novel long-lived transgenic mouse models of Gba deficiency, along with a subchronic conduritol-ß-epoxide (CBE) exposure paradigm. All three murine models revealed striking glial activation within nigrostriatal pathways, accompanied by abnormal α-synuclein accumulation. Importantly, the CBE-induced, pharmacological Gaucher mouse model replicated this change in dopamine neurotransmission, revealing a markedly reduced evoked striatal dopamine release (approximately 2-fold) that indicates synaptic dysfunction. Other changes in synaptic plasticity markers, including microRNA profile and a 24.9% reduction in post-synaptic density size, were concomitant with diminished evoked dopamine release following CBE exposure. These studies afford new insights into the mechanisms underlying the Parkinson's-Gaucher disease connection, and into the physiological impact of related abnormal α-synuclein accumulation and neuroinflammation on nigrostriatal dopaminergic neurotransmission. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Diverse metastable structures formed by small oligomers of α-synuclein probed by force spectroscopy.

    Directory of Open Access Journals (Sweden)

    Krishna Neupane

    Full Text Available Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson's disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.

  9. Ab initio alpha-alpha scattering

    CERN Document Server

    Elhatisari, Serdar; Rupak, Gautam; Epelbaum, Evgeny; Krebs, Hermann; Lähde, Timo A; Luu, Thomas; Meißner, Ulf-G

    2015-01-01

    Processes involving alpha particles and alpha-like nuclei comprise a major part of stellar nucleosynthesis and hypothesized mechanisms for thermonuclear supernovae. In an effort towards understanding alpha processes from first principles, we describe in this letter the first ab initio calculation of alpha-alpha scattering. We use lattice effective field theory to describe the low-energy interactions of nucleons and apply a technique called the adiabatic projection method to reduce the eight-body system to an effective two-cluster system. We find good agreement between lattice results and experimental phase shifts for S-wave and D-wave scattering. The computational scaling with particle number suggests that alpha processes involving heavier nuclei are also within reach in the near future.

  10. Oligomeric α-synuclein and β-amyloid variants as potential biomarkers for Parkinson's and Alzheimer's diseases.

    Science.gov (United States)

    Williams, Stephanie M; Schulz, Philip; Sierks, Michael R

    2016-01-01

    Oligomeric forms of α-synuclein and β-amyloid are toxic protein variants that are thought to contribute to the onset and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. The detection of toxic variants in human cerebrospinal fluid (CSF) and blood has great promise for facilitating early and accurate diagnoses of these devastating diseases. Two hurdles that have impeded the use of these protein variants as biomarkers are the availability of reagents that can bind the different variants and a sensitive assay to detect their very low concentrations. We previously isolated antibody-based reagents that selectively bind two different oligomeric variants of α-synuclein and two of β-amyloid, and developed a phage-based capture enzyme-linked immunosorbent assay (ELISA) with subfemtomolar sensitivity to quantify their presence. Here, we used these reagents to show that these oligomeric α-synuclein variants are preferentially present in PD brain tissue, CSF and serum, and that the oligomeric β-amyloid variants are preferentially present in AD brain tissue, CSF, and serum. Some AD samples also had α-synuclein pathology and some PD samples also had β-amyloid pathology, and, very intriguingly, these PD cases also had a history of dementia. Detection of different oligomeric α-synuclein and β-amyloid species is an effective method for identifying tissue, CSF and sera from PD and AD samples, respectively, and samples that also contained early stages of other protein pathologies, indicating their potential value as blood-based biomarkers for neurodegenerative diseases.

  11. Roles of autophagy in MPP+-induced neurotoxicity in vivo: the involvement of mitochondria and α-synuclein aggregation.

    Directory of Open Access Journals (Sweden)

    Kai-Chih Hung

    Full Text Available Macroautophagy (also known as autophagy is an intracellular self-eating mechanism and has been proposed as both neuroprotective and neurodestructive in the central nervous system (CNS neurodegenerative diseases. In the present study, the role of autophagy involving mitochondria and α-synuclein was investigated in MPP+ (1-methyl-4-phenylpyridinium-induced oxidative injury in chloral hydrate-anesthetized rats in vivo. The oxidative mechanism underlying MPP+-induced neurotoxicity was identified by elevated lipid peroxidation and heme oxygenase-1 levels, a redox-regulated protein in MPP+-infused substantia nigra (SN. At the same time, MPP+ significantly increased LC3-II levels, a hallmark protein of autophagy. To block MPP+-induced autophagy in rat brain, Atg7siRNA was intranigrally infused 4 d prior to MPP+ infusion. Western blot assay showed that in vivo Atg7siRNA transfection not only reduced Atg7 levels in the MPP+-infused SN but attenuated MPP+-induced elevation in LC3-II levels, activation of caspase 9 and reduction in tyrosine hydroxylase levels, indicating that autophagy is pro-death. The immunostaining study demonstrated co-localization of LC3 and succinate dehydrogenase (a mitochondrial complex II as well as LC3 and α-synuclein, suggesting that autophagy may engulf mitochondria and α-synuclein. Indeed, in vivo Atg7siRNA transfection mitigated MPP+-induced reduction in cytochrome c oxidase. In addition, MPP+-induced autophagy differentially altered the α-synuclein aggregates in the infused SN. In conclusion, autophagy plays a prodeath role in the MPP+-induced oxidative injury by sequestering mitochondria in the rat brain. Moreover, our data suggest that the benefits of autophagy depend on the levels of α-synuclein aggregates in the nigrostriatal dopaminergic system of the rat brain.

  12. Nitrate contamination of groundwater and its countermeasures

    Energy Technology Data Exchange (ETDEWEB)

    Mitamura, Hisayoshi [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2003-03-01

    The inevitable increases of food production and energy consumption with an increase in world population become main causes of an increase of nitrate load to the environment. Although nitrogen is essential for the growth of animal and plant as a constituent element of protein, excessive nitrate load to the environment contaminates groundwater resources used as drinking water and leads to seriously adverse effects on the health of man and livestock. In order to clarify the problem of nitrate contamination of groundwater and search a new trend of technology development from the viewpoint of environment remediation and protection, the present paper has reviewed adverse effects of nitrate on human health, the actual state of nitrogen cycle, several kinds of nitrate sources, measures for reducing nitrate level, etc. (author)

  13. Interaction of neodymium nitrate with rubidium and cesium nitrates

    Energy Technology Data Exchange (ETDEWEB)

    Molodkin, A.K.; Odinets, Z.K.; S' ' edina, T.V.; Ivanova, T.N. (Universitet Druzhby Narodov, Moscow (USSR))

    1982-12-01

    The Rb/sub 2/Nd(NO/sub 3/)/sub 5/xH/sub 2/O (1) and Cs/sub 2/Nd(NO/sub 3/)/sub 5/xH/sub 2/O (2) new complexes are prepared. The crystals 1 are isotropic, of cubic crystal system, Ng=1.570+-0.002; 2 - Ng=1.582+-0.002; Csub(Np)=0-9 degrees, of low crystal system (syngony). The bands of coordinated nitrate group, the ..delta nu..=..nu../sub 4/(B/sub 2/)-..nu../sub 1/(A/sub 1/) splitting value is respectively equal to 225 and 230 cm/sup -1/ are present in the infrared absorption spectra of the compounds. The interplane distances and corresponding intensities for the 1, 2 and hexahydrate of neodymium nitrate are determined. Derivatograms of the compounds are recorded, the final products of the thermolysis are correspondingly RbNdO/sub 2/ and Nd/sub 2/O/sub 3/.

  14. Dual effects of α-synuclein on neurotoxicity induced by low dosage of rotenone are dependent on exposure time in dopaminergic neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    This study was designed to investigate the effects of α-synuclein on toxicity induced by long-term exposure to relatively low concentrations of rotenone.Compared with the control groups,the inhibition of cell viability which overexpressed α-synuclein(SH-SY5Y-Syn) improved after 1 and 2 weeks of rotenone treatment.The complex I activity was greater and the mitochondrial membrane swelling intensity was reduced after 1 and 2 weeks of treatment,which indicated that α-synuclein,at least in part,resists the rotenone-induced oxidative stress.The results indicate that α-synuclein has a dual effect on toxicity of rotenone according to exposure time in human SH-SY5Y cells.

  15. Copper Binding and Subsequent Aggregation of α-Synuclein Are Modulated by N-Terminal Acetylation and Ablated by the H50Q Missense Mutation.

    Science.gov (United States)

    Mason, Rebecca J; Paskins, Aimee R; Dalton, Caroline F; Smith, David P

    2016-08-30

    The Parkinson's disease-associated protein α-synuclein exhibits significant conformational heterogeneity. Bacterially expressed α-synuclein is known to bind to copper, resulting in the formation of aggregation-prone compact conformations. However, in vivo, α-synuclein undergoes acetylation at its N-terminus. Here the effect of this modification and the pathological H50Q mutation on copper binding and subsequent conformational transitions were investigated by electrospray ionization-ion mobility spectrometry-mass spectrometry. We demonstrate that acetylation perturbs the ability of α-synuclein to bind copper and that the H50Q missense mutation in the presence of N-terminal acetylation prevents copper binding. These modifications and mutations prevent the formation of the most compact conformations and inhibit copper-induced aggregation.

  16. KINETICS STUDY ON NITRATION OF METHYL RICINOLEATE

    OpenAIRE

    Abdullah, Abdullah; Triyono, Triyono; Trisunaryanti, Wega; Haryadi, Winarto

    2012-01-01

    Kinetics parameter values of methyl ricinoleate nitration (rate constant, reaction order and the rate of reaction) have been determined. Nitration was carried out with both concentrations of HNO3 and acetic anhydride in excess to the concentration of methyl ricinoleate. Thus, the kinetics parameter value was only affected by the concentration of methyl ricinoleate. Based on kinetic study conducted, it could be concluded that the nitration follows pseudo first-order, and the reaction rate for ...

  17. Nitrate leaching from Silage Maize

    OpenAIRE

    Hansen, Elly Møller; Eriksen, Jørgen

    2009-01-01

    During the last 20 years the area with maize in Denmark has increased dramatically and reached 163,000 ha in 2008. Silage maize is easy to grow, is a suitable fodder for cows and goes well with grass-clover in the diet. This means that silage maize is often found in crop rotations with grass-clover on sandy soils in western Denmark. The ploughing in of grass-clover fields poses a serious risk of increased nitrate leaching on a coarse sandy soil, even when carried out in spring. With increased...

  18. Trend Analyses of Nitrate in Danish Groundwater

    Science.gov (United States)

    Hansen, B.; Thorling, L.; Dalgaard, T.; Erlandsen, M.

    2012-04-01

    This presentation assesses the long-term development in the oxic groundwater nitrate concentration and nitrogen (N) loss due to intensive farming in Denmark. Firstly, up to 20-year time-series from the national groundwater monitoring network enable a statistically systematic analysis of distribution, trends and trend reversals in the groundwater nitrate concentration. Secondly, knowledge about the N surplus in Danish agriculture since 1950 is used as an indicator of the potential loss of N. Thirdly, groundwater recharge CFC (Chlorofluorocarbon) age determination allows linking of the first two dataset. The development in the nitrate concentration of oxic groundwater clearly mirrors the development in the national agricultural N surplus, and a corresponding trend reversal is found in groundwater. Regulation and technical improvements in the intensive farming in Denmark have succeeded in decreasing the N surplus by 40% since the mid 1980s while at the same time maintaining crop yields and increasing the animal production of especially pigs. Trend analyses prove that the youngest (0-15 years old) oxic groundwater shows more pronounced significant downward nitrate trends (44%) than the oldest (25-50 years old) oxic groundwater (9%). This amounts to clear evidence of the effect of reduced nitrate leaching on groundwater nitrate concentrations in Denmark. Are the Danish groundwater monitoring strategy obtimal for detection of nitrate trends? Will the nitrate concentrations in Danish groundwater continue to decrease or are the Danish nitrate concentration levels now appropriate according to the Water Framework Directive?

  19. Measurement of isoprene nitrates by GCMS

    Science.gov (United States)

    Mills, Graham P.; Hiatt-Gipson, Glyn D.; Bew, Sean P.; Reeves, Claire E.

    2016-09-01

    According to atmospheric chemistry models, isoprene nitrates play an important role in determining the ozone production efficiency of isoprene; however this is very poorly constrained through observations as isoprene nitrates have not been widely measured. Measurements have been severely restricted largely due to a limited ability to measure individual isoprene nitrate isomers. An instrument based on gas chromatography/mass spectrometry (GCMS) and the associated calibration methods are described for the speciated measurements of individual isoprene nitrate isomers. Five of the primary isoprene nitrates which formed in the presence of NOx by reaction of isoprene with the hydroxyl radical (OH) in the Master Chemical Mechanism are identified using known isomers on two column phases and are fully separated on the Rtx-200 column. Three primary isoprene nitrates from the reaction of isoprene with the nitrate radical (NO3) are identified after synthesis from the already identified analogous hydroxy nitrate. A Tenax adsorbent-based trapping system allows the analysis of the majority of the known hydroxy and carbonyl primary isoprene nitrates, although not the (1,2)-IN isomer, under field-like levels of humidity and showed no impact from typical ambient concentrations of NOx and ozone.

  20. Nitration of Phenol Catalyzed by Horseradish Peroxidase

    Institute of Scientific and Technical Information of China (English)

    DAI Rong-ji; HUANG Hui; TONG Bin; XIAO Sheng-yuan

    2007-01-01

    Horseradish peroxidase, an acidic peroxidase from the horseradish, is one of the most important enzymes as analytical reagent.The enzymatic nitration of phenol by oxidation of nitrite was studied using horseradish peroxidase in the presence of H2O2.The results showed that nitration occur at 2- and 4- positions of phenol.There were also minor products of hydroquinone and catechol.The influence of various reaction parameters, including pH, organic solvent, and concentration of H2O2, on nitration products were discussed.The best nitration pH was 7.0, and H2O2 should be added to the reaction mixture slowly.

  1. Yttrium Nitrate mediated Nitration of Phenols at room temperature in Glacial Acetic acid

    Indian Academy of Sciences (India)

    MOHABUL A MONDAL; DBASHIS MANDAL; KANCHAN MITRA

    2017-01-01

    Rapid nitration of electron rich phenols using Y(NO₃)₃.6H₂O in glacial acetic acid at room temperature was observed with good yield. The method allows nitration of phenols without oxidation, and isolation of nitration product in a rapid and simple way. The described method is selective for phenols.

  2. Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport[W][OA

    Science.gov (United States)

    Wang, Ya-Yun; Tsay, Yi-Fang

    2011-01-01

    This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport. PMID:21571952

  3. Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport.

    Science.gov (United States)

    Wang, Ya-Yun; Tsay, Yi-Fang

    2011-05-01

    This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport.

  4. Challenges with nitrate therapy and nitrate tolerance: prevalence, prevention, and clinical relevance.

    Science.gov (United States)

    Thadani, Udho

    2014-08-01

    Nitrate therapy has been an effective treatment for ischemic heart disease for over 100 years. The anti-ischemic and exercise-promoting benefits of sublingually administered nitrates are well established. Nitroglycerin is indicated for the relief of an established attack of angina and for prophylactic use, but its effects are short lived. In an effort to increase the duration of beneficial effects, long-acting orally administered and topical applications of nitrates have been developed; however, following their continued or frequent daily use, patients soon develop tolerance to these long-acting nitrate preparations. Once tolerance develops, patients begin losing the protective effects of the long-acting nitrate therapy. By providing a nitrate-free interval, or declining nitrate levels at night, one can overcome or reduce the development of tolerance, but cannot provide 24-h anti-anginal and anti-ischemic protection. In addition, patients may be vulnerable to occurrence of rebound angina and myocardial ischemia during periods of absent nitrate levels at night and early hours of the morning, and worsening of exercise capacity prior to the morning dose of the medication. This has been a concern with nitroglycerin patches but not with oral formulations of isosorbide-5 mononitrates, and has not been adequately studied with isosorbide dinitrate. This paper describes problems associated with nitrate tolerance, reviews mechanisms by which nitrate tolerance and loss of efficacy develop, and presents strategies to avoid nitrate tolerance and maintain efficacy when using long-acting nitrate formulations.

  5. Impairment of mitochondria dynamics by human A53T α-synuclein and rescue by NAP (davunetide) in a cell model for Parkinson's disease.

    Science.gov (United States)

    Melo, T Q; van Zomeren, K C; Ferrari, M F R; Boddeke, H W G M; Copray, J C V M

    2017-03-01

    The formation of oligomers and aggregates of overexpressed or mutant α-synuclein play a role in the degeneration of dopaminergic neurons in Parkinson's disease by causing dysfunction of mitochondria, reflected in their disturbed mobility and production of ROS. The mode of action and mechanisms underlying this mitochondrial impairment is still unclear. We have induced stable expression of wild-type, A30P or A53T α-synuclein in neuronally differentiated SH-SY5Y neuroblastoma cells and studied anterograde and retrograde mitochondrial trafficking in this cell model for Parkinson's disease. In contrast to wild-type and A30P, A53T α-synuclein significantly inhibited mitochondrial trafficking, at first retrogradely and in a later stage anterogradely. Accordingly, A53T α-synuclein also caused the highest increase in ROS production in the dysmobilized mitochondria in comparison to wild-type or A30P α-synuclein. Treatment with NAP, the eight amino acid peptide identified as the active component of activity-dependent neuroprotective protein (ADNP), completely annihilated the adverse effects of A53T on mitochondrial dynamics. Our results reveal that A53T α-synuclein (oligomers or aggregates) leads to the inhibition of mitochondrial trafficking, which can be rescued by NAP, suggesting the involvement of microtubule disruption in the pathophysiology of Parkinson's disease.

  6. Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein by Small Molecules as a Potential Therapeutic Strategy for Parkinson’s Disease

    Science.gov (United States)

    Tóth, Gergely; Gardai, Shyra J.; Zago, Wagner; Bertoncini, Carlos W.; Cremades, Nunilo; Roy, Susan L.; Tambe, Mitali A.; Rochet, Jean-Christophe; Galvagnion, Celine; Skibinski, Gaia; Finkbeiner, Steven; Bova, Michael; Regnstrom, Karin; Chiou, San-San; Johnston, Jennifer; Callaway, Kari; Anderson, John P.; Jobling, Michael F.; Buell, Alexander K.; Yednock, Ted A.; Knowles, Tuomas P. J.; Vendruscolo, Michele; Christodoulou, John; Dobson, Christopher M.; Schenk, Dale; McConlogue, Lisa

    2014-01-01

    The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson’s and Alzheimer’s diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson’s disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson’s disease and related conditions. PMID:24551051

  7. Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Gergely Tóth

    Full Text Available The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228, that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.

  8. Faddeev calculation of 3 alpha and alpha alpha Lambda systems using alpha alpha resonating-group method kernel

    CERN Document Server

    Fujiwara, Y; Kohno, M; Suzuki, Y; Baye, D; Sparenberg, J M

    2004-01-01

    We carry out Faddeev calculations of three-alpha (3 alpha) and two-alpha plus Lambda (alpha alpha Lambda) systems, using two-cluster resonating-group method kernels. The input includes an effective two-nucleon force for the alpha alpha resonating-group method and a new effective Lambda N force for the Lambda alpha interaction. The latter force is a simple two-range Gaussian potential for each spin-singlet and triplet state, generated from the phase-shift behavior of the quark-model hyperon-nucleon interaction, fss2, by using an inversion method based on supersymmetric quantum mechanics. Owing to the exact treatment of the Pauli-forbidden states between the clusters, the present three-cluster Faddeev formalism can describe the mutually related, alpha alpha, 3 alpha and alpha alpha Lambda systems, in terms of a unique set of the baryon-baryon interactions. For the three-range Minnesota force which describes the alpha alpha phase shifts quite accurately, the ground-state and excitation energies of 9Be Lambda are...

  9. Evaluation of nitrates in albanian wines

    Directory of Open Access Journals (Sweden)

    Ariola Morina

    2013-05-01

    Full Text Available Nitrates are important compounds in nature but not desirable if they are present in wine at increased amount. The high level of nitrate is attributed to the use of nitrogen fertilizers in the vineyards. Method of the reactive Gries I and Gries II was used for the determination of nitrates in wine. There were analyzed 45 white wines and 55 red wines produced in 2008 – 2010, as well as wines produced from Albanian grape varieties Shesh i Bardhë and Shesh i Zi in 2009 and 2010, as an authentic wines evidence with denominated origin. From the results of analyses was observed that, in 51 % of white wines was found that the content of nitrates were less than 5 mg/l, in 46% of them the nitrates level goes up to 10 mg/l and only in 3 % of them the amount of nitrates is up to 12 mg/l. None of white wine samples have the content of nitrates over 20 mg/l. In this case there is no doubt for water addition during wine preparation. In regards of red wines, in 34% of them the amount of nitrates is up to 5 mg/l, in 30% of them up to 10 mg/l, while in 26% of them the amount of nitrates is 20 mg/l. Only 10 % of red wines have nitrates content over 20 mg/l which raise dubiety for falsified wines where water and sugar is added in the red marc. The level of nitrates in wines with denominated origin was under 20 mg/L.

  10. Regulation of nitrate assimilation in cyanobacteria.

    Science.gov (United States)

    Ohashi, Yoshitake; Shi, Wei; Takatani, Nobuyuki; Aichi, Makiko; Maeda, Shin-ichi; Watanabe, Satoru; Yoshikawa, Hirofumi; Omata, Tatsuo

    2011-02-01

    Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

  11. Vitamins K interact with N-terminus α-synuclein and modulate the protein fibrillization in vitro. Exploring the interaction between quinones and α-synuclein.

    Science.gov (United States)

    da Silva, Fernanda Luna; Coelho Cerqueira, Eduardo; de Freitas, Mônica Santos; Gonçalves, Daniela Leão; Costa, Lilian Terezinha; Follmer, Cristian

    2013-01-01

    In the last decades, a series of compounds, including quinones and polyphenols, has been described as having anti-fibrillogenic action on α-synuclein (α-syn) whose aggregation is associated to the pathogenesis of Parkinson's disease (PD). Most of these molecules act as promiscuous anti-amyloidogenic agents, interacting with the diverse amyloidogenic proteins (mostly unfolded) through non-specific hydrophobic interactions. Herein we investigated the effect of the vitamins K (phylloquinone, menaquinone and menadione), which are 1,4-naphthoquinone (1,4-NQ) derivatives, on α-syn aggregation, comparing them with other anti-fibrillogenic molecules such as quinones, polyphenols and lipophilic vitamins. Vitamins K delayed α-syn fibrillization in substoichiometric concentrations, leading to the formation of short, sheared fibrils and amorphous aggregates, which are less prone to produce leakage of synthetic vesicles. In seeding conditions, menadione and 1,4-NQ significantly inhibited fibrils elongation, which could be explained by their ability to destabilize preformed fibrils of α-syn. Bidimensional NMR experiments indicate that a specific site at the N-terminal α-syn (Gly31/Lys32) is involved in the interaction with vitamins K, which is corroborated by previous studies suggesting that Lys is a key residue in the interaction with quinones. Together, our data suggest that 1,4-NQ, recently showed up by our group as a potential scaffold for designing new monoamine oxidase inhibitors, is also capable to modulate α-syn fibrillization in vitro.

  12. Alpha Thalassemia (For Parents)

    Science.gov (United States)

    ... Kids to Be Smart About Social Media Alpha Thalassemia KidsHealth > For Parents > Alpha Thalassemia Print A A ... Complications Symptoms Diagnosis Treatment en español Alfa talasemia Thalassemias Thalassemias are a group of blood disorders that ...

  13. Nitrate reduction functional genes and nitrate reduction potentials persist in deeper estuarine sediments. Why?

    Directory of Open Access Journals (Sweden)

    Sokratis Papaspyrou

    Full Text Available Denitrification and dissimilatory nitrate reduction to ammonium (DNRA are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the

  14. Nitrate Reduction Functional Genes and Nitrate Reduction Potentials Persist in Deeper Estuarine Sediments. Why?

    Science.gov (United States)

    Papaspyrou, Sokratis; Smith, Cindy J.; Dong, Liang F.; Whitby, Corinne; Dumbrell, Alex J.; Nedwell, David B.

    2014-01-01

    Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the sediment column. This

  15. AlphaACT

    Science.gov (United States)

    2014-07-20

    sulfate Cerium trioxide Cesium Cesium hydroxide Chlorine Chlorobenzyhdene malononitrile, o- Chlorodecane, 1- Chlorodiphenylarsine Chloroform...Ethylbenzaldehyde Ethylene dimethacrylate Ethylene glycol Ethylmercury chloride Europium oxide Ferric nitrate Ferric sulfate Ferrous ammonium

  16. The Sybtraps: control of synaptobrevin traffic by synaptophysin, α-synuclein and AP-180.

    Science.gov (United States)

    Gordon, Sarah L; Cousin, Michael A

    2014-03-01

    Synaptobrevin II (sybII) is a key fusogenic molecule on synaptic vesicles (SVs) therefore the active maintenance of both its conformation and location in sufficient numbers on this organelle is critical in both mediating and sustaining neurotransmitter release. Recently three proteins have been identified having key roles in the presentation, trafficking and retrieval of sybII during the fusion and endocytosis of SVs. The nerve terminal protein α-synuclein catalyses sybII entry into SNARE complexes, whereas the monomeric adaptor protein AP-180 is required for sybII retrieval during SV endocytosis. Overarching these events is the tetraspan SV protein synaptophysin, which is a major sybII interaction partner on the SV. This review will evaluate recent studies to propose working models for the control of sybII traffic by synaptophysin and other Sybtraps (sybII trafficking partners) and suggest how dysfunction in sybII traffic may contribute to human disease.

  17. 5-Fluorotryptophan as a Dual NMR and Fluorescent Probe of α-Synuclein

    Science.gov (United States)

    Pfefferkorn, Candace M.; Lee, Jennifer C.

    2012-01-01

    i. Summary Analysis of conventional proton nuclear magnetic resonance (NMR) experiments on intrinsically disordered proteins (IDPs) is challenging because of the highly flexible and multiple rapidly exchanging conformations typifying this class of proteins. One method to circumvent some of these difficulties is to incorporate non-native fluorine (19F) nuclei at specific sites within the polypeptide. 19F NMR is particularly suitable for characterization of unfolded structures because 19F chemical shifts are highly sensitive to local environments and conformations. Furthermore, the incorporation of fluorine analogs of fluorescent amino acids such as 5-fluoro-tryptophan (5FW) allows for complementary studies of protein microenvironment via fluorescence spectroscopy. Herein we describe methods to produce, purify, characterize, and perform steady-state fluorescence and 1-D NMR experiments on 5FW analogues of the IDP α-synuclein. PMID:22760321

  18. α-Synuclein rs356219 polymorphisms in patients with Gaucher disease and Parkinson disease.

    Science.gov (United States)

    Altarescu, Gheona; Ioscovich, Daniel; Alcalay, Roy N; Zimran, Ari; Elstein, Deborah

    2014-09-19

    Mutations in β-glucocerebrosidase, the genetic defect in Gaucher disease (GD), are an important susceptibility factor for Parkinson disease (PD). A PD effector is α-synuclein (SNCA) hypothesized to selectively interact with β-glucocerebrosidase under lysosomal conditions. SNCA polymorphism rs356219 may be associated with early-age-onset PD, common among patients with GD+PD. The objective of this study was to ascertain rs356219 genotypes of GD+PD patients. All GD+PD patients at our Gaucher referral clinic were asked to participate. A GD-only sex-, age-, GD genotype-, and enzyme therapy (ERT)-matched control was found for each GD+PD participant. Student's t-test was used (p-value 500 adult GD patients in our clinic. Nonetheless, as a foray into potential genetic GD susceptibility for a synucleinopathy, this study suggests the need for collaboration to achieve larger sample size. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Computational insight into nitration of human myoglobin.

    Science.gov (United States)

    Lin, Ying-Wu; Shu, Xiao-Gang; Du, Ke-Jie; Nie, Chang-Ming; Wen, Ge-Bo

    2014-10-01

    Protein nitration is an important post-translational modification regulating protein structure and function, especially for heme proteins. Myoglobin (Mb) is an ideal protein model for investigating the structure and function relationship of heme proteins. With limited structural information available for nitrated heme proteins from experiments, we herein performed a molecular dynamics study of human Mb with successive nitration of Tyr103, Tyr146, Trp7 and Trp14. We made a detailed comparison of protein motions, intramolecular contacts and internal cavities of nitrated Mbs with that of native Mb. It showed that although nitration of both Tyr103 and Tyr146 slightly alters the local conformation of heme active site, further nitration of both Trp7 and Trp14 shifts helix A apart from the rest of protein, which results in altered internal cavities and forms a water channel, representing an initial stage of Mb unfolding. The computational study provides an insight into the nitration of heme proteins at an atomic level, which is valuable for understanding the structure and function relationship of heme proteins in non-native states by nitration. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Bacterial nitrate assimilation: gene distribution and regulation.

    Science.gov (United States)

    Luque-Almagro, Víctor M; Gates, Andrew J; Moreno-Vivián, Conrado; Ferguson, Stuart J; Richardson, David J; Roldán, M Dolores

    2011-12-01

    In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism.

  1. Dietary Nitrate, Nitric Oxide, and Cardiovascular Health.

    Science.gov (United States)

    Bondonno, Catherine P; Croft, Kevin D; Hodgson, Jonathan M

    2016-09-09

    Emerging evidence strongly suggests that dietary nitrate, derived in the diet primarily from vegetables, could contribute to cardiovascular health via effects on nitric oxide (NO) status. NO plays an essential role in cardiovascular health. It is produced via the classical L-arginine-NO-synthase pathway and the recently discovered enterosalivary nitrate-nitrite-NO pathway. The discovery of this alternate pathway has highlighted dietary nitrate as a candidate for the cardioprotective effect of a diet rich in fruit and vegetables. Clinical trials with dietary nitrate have observed improvements in blood pressure, endothelial function, ischemia-reperfusion injury, arterial stiffness, platelet function, and exercise performance with a concomitant augmentation of markers of NO status. While these results are indicative of cardiovascular benefits with dietary nitrate intake, there is still a lingering concern about nitrate in relation to methemoglobinemia, cancer, and cardiovascular disease. It is the purpose of this review to present an overview of NO and its critical role in cardiovascular health; to detail the observed vascular benefits of dietary nitrate intake through effects on NO status as well as to discuss the controversy surrounding the possible toxic effects of nitrate.

  2. 4-Methoxy-N,N′-diphenylbenzamidinium nitrate

    Directory of Open Access Journals (Sweden)

    Renata S. Silva

    2016-09-01

    Full Text Available The asymmetric unit of the title salt N,N′-diphenyl-4-methoxybenzamidinium nitrate, C20H19N2O+·NO3−, comprises two independent N,N′-diphenyl-4-methoxybenzamidinium cations and two nitrate anions. The crystal structure features N—H...O hydrogen bonds and C—H...O contacts responsible for the packing.

  3. The Alpha Antihydrogen Experiment

    Science.gov (United States)

    Madsen, N.; Andresen, G.; Bertsche, W.; Boston, A.; Bowe, P. D.; Butler, E.; Cesar, C. L.; Chapman, S.; Charlton, M.; Chartier, M.; Fajans, J.; Funakoshi, R.; Gill, D. R.; Hangst, J. S.; Hardy, W. N.; Hayano, R. S.; Hayden, M.; Hydomako, R.; Jenkins, M. J.; Jørgensen, L. V.; Kurchaninov, L.; Nolan, P.; Olchanski, K.; Olin, A.; Page, R. D.; Povilus, A.; Robicheaux, F.; Sarid, E.; Seif El Nasr, S.; Silveira, D. M.; Storey, J. W.; Thompson, R. I.; van der Werf, D. P.; Wurtele, J. S.; Yamazaki, Y.

    2008-03-01

    ALPHA is a new experiment at the CERN Antiproton Decelerator (AD). The short term goal of ALPHA is trapping of cold antihydrogen, with the long term goal of conducting precise spectroscopic comparisons of hydrogen and antihydrogen. Here we present the current status of ALPHA and the physics considerations and results leading to its design as well as recent progress towards trapping.

  4. Copper(I/II), α/β-Synuclein and Amyloid-β: Menage à Trois?

    Science.gov (United States)

    De Ricco, Riccardo; Valensin, Daniela; Dell'Acqua, Simone; Casella, Luigi; Hureau, Christelle; Faller, Peter

    2015-11-02

    Copper binding to α-synuclein (aS) and to amyloid-β (Ab) has been connected to Parkinson's and Alzheimer's disease (AD), respectively, because Cu ions can modulate the peptide aggregation, and these Cu ⋅ peptide complexes can catalyse the production of reactive oxygen species (ROS). In a significant proportion of AD brains, aggregation of aS and Ab has been detected, and it was proposed that Ab and aS interact with each other. Thus, we investigated the potential interactions of Ab and aS through their binding of copper(I) and copper(II). Additionally, β-synuclein (bS) was investigated, due to its additional methionine residue, a potential Cu(I) ligand. We found that: 1) the peptides containing the Cu-binding domains Ab1-16, aS1-15 and bS1-15 have similar affinities towards Cu(II) and towards Cu(I), with Ab1-16 being slightly stronger, 2) in the case of Cu(I), the additional Met residue in bS1-15 increased the affinity slightly, 3) the exchange of Cu(I/II) between the two peptides is rapid (≤ ms), 4) a/bS1-15 and Ab1-16 form a heterodimeric complex with Cu(II), 5) Cu(I) probably promotes a transient ternary complex, 6) the different Cu(I/II) coordination of Ab1-16, aS1-15 and bS1-15 impacts the capacity to produce ROS and to oxidise catechol, and 7) when Ab1-16, aS1-15 and Cu are present, the ROS production more closely resembles that by Ab1-16. The work gives insights into the coordination chemistry of these related peptides, and the relevance of coordination differences, the ternary complex and ROS production are discussed.

  5. Nascent histamine induces α-synuclein and caspase-3 on human cells

    Energy Technology Data Exchange (ETDEWEB)

    Caro-Astorga, Joaquín; Fajardo, Ignacio; Ruiz-Pérez, María Victoria; Sánchez-Jiménez, Francisca; Urdiales, José Luis, E-mail: jlurdial@uma.es

    2014-09-05

    Highlights: • Nascent histamine alters cyclin expression pattern. • Nascent histamine increases expression of α-synuclein. • Nascent histamine activates caspase-3. - Abstract: Histamine (Hia) is the most multifunctional biogenic amine. It is synthetized by histidine decarboxylase (HDC) in a reduced set of mammalian cell types. Mast cells and histaminergic neurons store Hia in specialized organelles until the amine is extruded by exocytosis; however, other immune and cancer cells are able to produce but not store Hia. The intracellular effects of Hia are still not well characterized, in spite of its physiopathological relevance. Multiple functional relationships exist among Hia metabolism/signaling elements and those of other biogenic amines, including growth-related polyamines. Previously, we obtained the first insights for an inhibitory effect of newly synthetized Hia on both growth-related polyamine biosynthesis and cell cycle progression of non-fully differentiated mammalian cells. In this work, we describe progress in this line. HEK293 cells were transfected to express active and inactive versions of GFP-human HDC fusion proteins and, after cell sorting by flow cytometry, the relative expression of a large number of proteins associated with cell signaling were measured using an antibody microarray. Experimental results were analyzed in terms of protein–protein and functional interaction networks. Expression of active HDC induced a cell cycle arrest through the alteration of the levels of several proteins such as cyclin D1, cdk6, cdk7 and cyclin A. Regulation of α-synuclein and caspase-3 was also observed. The analyses provide new clues on the molecular mechanisms underlying the regulatory effects of intracellular newly synthetized Hia on cell proliferation/survival, cell trafficking and protein turnover. This information is especially interesting for emergent and orphan immune and neuroinflammatory diseases.

  6. Phenolic compounds prevent the oligomerization of α-synuclein and reduce synaptic toxicity.

    Science.gov (United States)

    Takahashi, Ryoichi; Ono, Kenjiro; Takamura, Yusaku; Mizuguchi, Mineyuki; Ikeda, Tokuhei; Nishijo, Hisao; Yamada, Masahito

    2015-09-01

    Lewy bodies, mainly composed of α-synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low-order αS oligomers are neurotoxic and critical species in the pathogenesis of α-synucleinopathies. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo-induced cross-linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N-terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long-term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds on α-synuclein (αS) oligomerization. Phenolic compounds, especially Myricetin (Myr) and Rosmarinic acid (RA), inhibited αS oligomerization and secondary structure conversion. Myr and RA ameliorated αS synaptic toxicity on the experiment of long-term potentiation. Our results suggest that Myr and RA prevent αS aggregation process and reduce the neurotoxicity of αS oligomers. Phenolic compounds are good

  7. A new method for quantitative immunoblotting of endogenous α-synuclein.

    Directory of Open Access Journals (Sweden)

    Andrew J Newman

    Full Text Available β-Sheet-rich aggregates of α-synuclein (αSyn are the hallmark neuropathology of Parkinson's disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells--unfolded monomer or α-helically folded oligomer--is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.

  8. Nitrate reduction in an unconfined sandy aquifer

    DEFF Research Database (Denmark)

    Postma, Diederik Jan; Boesen, Carsten; Kristiansen, Henning;

    1991-01-01

    Nitrate distribution and reduction processes were investigated in an unconfined sandy aquifer of Quaternary age. Groundwater chemistry was studied in a series of eight multilevel samplers along a flow line, deriving water from both arable and forested land. Results show that plumes of nitrate...... processes of O2 and NO3- occur at rates that are fast compared to the rate of downward water transport. Nitrate-contaminated groundwater contains total contents of dissolved ions that are two to four times higher than in groundwater derived from the forested area. The persistence of the high content...... of total dissolved ions in the NO3- free anoxic zone indicates the downward migration of contaminants and that active nitrate reduction is taking place. Nitrate is apparently reduced to N2 because both nitrite and ammonia are absent or found at very low concentrations. Possible electron donors...

  9. Glucocerebrosidase deficiency accelerates the accumulation of proteinase K-resistant α-synuclein and aggravates neurodegeneration in a Drosophila model of Parkinson's disease.

    Science.gov (United States)

    Suzuki, Mari; Fujikake, Nobuhiro; Takeuchi, Toshihide; Kohyama-Koganeya, Ayako; Nakajima, Kazuki; Hirabayashi, Yoshio; Wada, Keiji; Nagai, Yoshitaka

    2015-12-01

    Alpha-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Recent multicenter genetic studies have revealed that mutations in the glucocerebrosidase 1 (GBA1) gene, which are responsible for Gaucher's disease, are strong risk factors for PD and DLB. However, the mechanistic link between the functional loss of glucocerebrosidase (GCase) and the toxicity of αSyn in vivo is not fully understood. In this study, we employed Drosophila models to examine the effect of GCase deficiency on the neurotoxicity of αSyn and its molecular mechanism. Behavioral and histological analyses showed that knockdown of the Drosophila homolog of GBA1 (dGBA1) exacerbates the locomotor dysfunction, loss of dopaminergic neurons and retinal degeneration of αSyn-expressing flies. This phenotypic aggravation was associated with the accumulation of proteinase K (PK)-resistant αSyn, rather than with changes in the total amount of αSyn, raising the possibility that glucosylceramide (GlcCer), a substrate of GCase, accelerates the misfolding of αSyn. Indeed, in vitro experiments revealed that GlcCer directly promotes the conversion of recombinant αSyn into the PK-resistant form, representing a toxic conformational change. Similar to dGBA1 knockdown, knockdown of the Drosophila homolog of β-galactosidase (β-Gal) also aggravated locomotor dysfunction of the αSyn flies, and its substrate GM1 ganglioside accelerated the formation of PK-resistant αSyn. Our findings suggest that the functional loss of GCase or β-Gal promotes the toxic conversion of αSyn via aberrant interactions between αSyn and their substrate glycolipids, leading to the aggravation of αSyn-mediated neurodegeneration.

  10. Sedimentary nitrate reduction and its effect on the N-isotopic composition of oceanic nitrate

    Science.gov (United States)

    Lehmann, M. F.; Sigman, D. M.; McCorkle, D. C.

    2005-12-01

    A prerequisite for assessing denitrification fluxes in a specific environment using water column nitrate N isotope ratios is the knowledge of the expressed N isotope effects of water column and/or benthic denitrification in this environment. Here, we aim at assessing the effects of benthic nitrogen cycling on the N isotopic composition of the oceanic nitrate pool in deep-sea sediments, which are believed to harbour a large portion of the global benthic denitrification. We report 15N/14N ratios of pore water nitrate in pelagic sediments from the deep Bering Sea, where benthic nitrate reduction has previously been identified as a significant sink of fixed nitrogen. Porewater profiles from multicores indicate strong 15N enrichment in porewater nitrate at all stations, as one goes deeper in the sediments and nitrate concentrations decrease (δ15N generally reached 25-35‰). Our data are consistent with variable biological isotope effect (ɛ) for dissimilatory nitrate reduction ranging between 13 to 30 ‰. A one-dimensional diffusion-reaction model including organic matter degradation, nitrification, and denitrification indicates that, although denitrification leads to a pore water nitrate pool that is enriched in 15N, N isotope fractionation is poorly expressed at the scale of sediment-water nitrate exchange, independent of whether sediments are a net sink or a net source of nitrate. The apparent nitrate isotope effect of sedimentary denitrification on nitrate in overlying waters is generally below 2‰, as a result of diffusive transport limitation into, and within, the sediments and/or the production of light nitrate during nitrification. Thus, our data suggest that the low expressed isotope effect of benthic denitrification observed previously in reactive shelf sediments also applies to deep-sea sediments. However, where ammonium fluxes out of the sediments, it is enriched in 15-N, and may ultimately lead to an N-isotopic enrichment of the water-column nitrate

  11. Trehalose Inhibits A53T Mutant α-Synuclein Overexpression and Neurotoxicity in Transduced PC12 Cells.

    Science.gov (United States)

    Zhao, Juan; Zhi, Xiuling; Pan, Luanfeng; Zhou, Ping

    2017-08-08

    Fibrillar accumulation of A53T mutant α-synuclein (A53T-AS) in Lewy bodies is a symptom of Parkinsonism. Inhibitions of the overexpression and fibrillar aggregation of α-synuclein (AS) in vivo could be a promising strategy for treating Parkinson's disease (PD). In this study, at concentrations lower than 1 mM, trehalose decreased the A53T-AS expression level in transduced PC12 cells. Although H₂O₂ and aluminum ions increased the expression level and neurotoxicity of A53T-AS in cells, proper trehalose concentrations inhibited the event. These studies adequately prove that trehalose at an appropriate dose would be potentially useful for PD treatment.

  12. Structural characterization of copper(II) binding to α-synuclein: Insights into the bioinorganic chemistry of Parkinson's disease

    OpenAIRE

    Rasia, Rodolfo M.; Carlos W Bertoncini; Marsh, Derek; Hoyer, Wolfgang; Cherny, Dmitry; Zweckstetter, Markus; Griesinger, Christian; Jovin, Thomas M.; Fernández, Claudio O

    2005-01-01

    The aggregation of α-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring r...

  13. Structural characterization of copper(II) binding to α-Synuclein: Insights into the bioinorganic chemistry of Parkinson's disease

    OpenAIRE

    Rasia, R.; BERTONCINI, C; Marsh, D; Hoyer, W.; Cherny, D; Zweckstetter, M.; Griesinger, C; Jovin, T.; Fernandez, C.

    2005-01-01

    The aggregation of α -synuclein (AS) is characteristic of Parkinson’s disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring ...

  14. A QUANTITATIVE STUDY OF α-SYNUCLEIN PATHOLOGY IN FIFTEEN CASES OF DEMENTIA ASSOCIATED WITH PARKINSON DISEASE

    OpenAIRE

    Armstrong, Richard A.; Kotzbauer, Paul T.; Perlmutter, Joel S.; Campbell, Meghan C.; Hurth, Kyle M; Schmidt, Robert E.; Cairns, Nigel J.

    2013-01-01

    The α-synuclein-immunoreactive pathology of dementia associated with Parkinson disease (DPD) comprises Lewy bodies (LB), Lewy neurites (LN), and Lewy grains (LG). The densities of LB, LN, LG together with vacuoles, neurons, abnormally enlarged neurons (EN), and glial cell nuclei were measured in fifteen cases of DPD. Densities of LN and LG were up to 19 and 70 times those of LB respectively, depending on region. Densities were significantly greater in amygdala, entorhinal cortex (EC), and sec...

  15. Downregulation of microRNA-497 is associated with upregulation of synuclein γ in patients with osteosarcoma

    OpenAIRE

    Wang, Liang; Gao, Hongwei; Gong, Ningji; Gong, Mingzhi

    2016-01-01

    The present study aimed to investigate the effects of microRNA (miRNA/miR)-497 expression levels on the expression levels of synuclein γ (SNCG) in serum samples, as well as osteosarcoma and lung-metastatic tissue samples, from patients with osteosarcoma. Between December 2010 and August 2013, fasting peripheral blood was collected from 36 patients with osteosarcoma for serum separation. In addition, osteosarcoma and lung metastatic tissues were resected from 15 osteosarcoma patients with lung...

  16. Parkinson disease: α-synuclein mutational screening and new clinical insight into the p.E46K mutation.

    Science.gov (United States)

    Pimentel, Márcia M G; Rodrigues, Fabíola C; Leite, Marco Antônio A; Campos Júnior, Mário; Rosso, Ana Lucia; Nicaretta, Denise H; Pereira, João S; Silva, Delson José; Della Coletta, Marcus V; Vasconcellos, Luiz Felipe R; Abreu, Gabriella M; Dos Santos, Jussara M; Santos-Rebouças, Cíntia B

    2015-06-01

    Amongst Parkinson's disease-causing genetic factors, missense mutations and genomic multiplications in the gene encoding α-synuclein are well established causes of the disease, although genetic data in populations with a high degree of admixture, such as the Brazilian one, are still scarce. In this study, we conducted a molecular screening of α-synuclein point mutations and copy number variation in the largest cohort of Brazilian patients with Parkinson's disease (n = 549) and also in twelve Portuguese and one Bolivian immigrants. Genomic DNA was isolated from peripheral blood leukocytes or saliva, and the mutational screening was performed by quantitative and qualitative real-time PCR. The only alteration identified was the p.E46K mutation in a 60-year-old man, born in Bolivia, with a familial history of autosomal dominant Parkinson's disease. This is the second family ever reported, in which this rare pathogenic mutation is segregating. The same mutation was firstly described ten years ago in a Spanish family with a neurodegenerative syndrome combining parkinsonism, dementia and visual hallucinations. The clinical condition of our proband reveals a less aggressive phenotype than previously described and reinforces that marked phenotypic heterogeneity is common among patients with Parkinson's disease, even among those carriers sharing the same mutation. Our findings add new insight into the preexisting information about α-synuclein p.E46K, improving our understanding about the endophenotypes associated to this mutation and corroborate that missense alterations and multiplications in α-synuclein are uncommon among Brazilian patients with Parkinson's disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. The determination of $\\alpha_s$ by the ALPHA collaboration

    CERN Document Server

    Bruno, Mattia

    2016-01-01

    We review the ALPHA collaboration strategy for obtaining the QCD coupling at high scale. In the three-flavor effective theory it avoids the use of perturbation theory at $\\alpha > 0.2$ and at the same time has the physical scales small compared to the cutoff $1/a$ in all stages of the computation. The result $\\Lambda_\\overline{MS}^{(3)}=332(14)$~MeV is translated to $\\alpha_\\overline{MS}(m_Z)=0.1179(10)(2)$ by use of (high order) perturbative relations between the effective theory couplings at the charm and beauty quark "thresholds". The error of this perturbative step is discussed and estimated as $0.0002$.

  18. Modeling nitrate removal in a denitrification bed.

    Science.gov (United States)

    Ghane, Ehsan; Fausey, Norman R; Brown, Larry C

    2015-03-15

    Denitrification beds are promoted to reduce nitrate load in agricultural subsurface drainage water to alleviate the adverse environmental effects associated with nitrate pollution of surface water. In this system, drainage water flows through a trench filled with a carbon media where nitrate is transformed into nitrogen gas under anaerobic conditions. The main objectives of this study were to model a denitrification bed treating drainage water and evaluate its adverse greenhouse gas emissions. Field experiments were conducted at an existing denitrification bed. Evaluations showed very low greenhouse gas emissions (mean N2O emission of 0.12 μg N m(-2) min(-1)) from the denitrification bed surface. Field experiments indicated that nitrate removal rate was described by Michaelis-Menten kinetics with the Michaelis-Menten constant of 7.2 mg N L(-1). We developed a novel denitrification bed model based on the governing equations for water flow and nitrate removal kinetics. The model evaluation statistics showed satisfactory prediction of bed outflow nitrate concentration during subsurface drainage flow. The model can be used to design denitrification beds with efficient nitrate removal which in turn leads to enhanced drainage water quality.

  19. Mutant LRRK2 toxicity in neurons depends on LRRK2 levels and synuclein but not kinase activity or inclusion bodies.

    Science.gov (United States)

    Skibinski, Gaia; Nakamura, Ken; Cookson, Mark R; Finkbeiner, Steven

    2014-01-08

    By combining experimental neuron models and mathematical tools, we developed a "systems" approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration.

  20. Mutant LRRK2 Toxicity in Neurons Depends on LRRK2 Levels and Synuclein But Not Kinase Activity or Inclusion Bodies

    Science.gov (United States)

    Skibinski, Gaia; Nakamura, Ken; Cookson, Mark R.

    2014-01-01

    By combining experimental neuron models and mathematical tools, we developed a “systems” approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration. PMID:24403142

  1. Photodegradation of Paracetamol in Nitrate Solution

    Science.gov (United States)

    Meng, Cui; Qu, Ruijuan; Liang, Jinyan; Yang, Xi

    2010-11-01

    The photodegradation of paracetamol in nitrate solution under simulated solar irradiation has been investigated. The degradation rates were compared by varying environmental parameters including concentrations of nitrate ion, humic substance and pH values. The quantifications of paracetamol were conducted by HPLC method. The results demonstrate that the photodegradation of paracetamol followed first-order kinetics. The photoproducts and intermediates of paracetamol in the presence of nitrate ions were identified by extensive GC-MS method. The photodegradation pathways involving. OH radicals as reactive species were proposed.

  2. Analytical Characterization of the Thorium Nitrate Stockpile

    Energy Technology Data Exchange (ETDEWEB)

    Mattus, CH

    2003-12-30

    For several years, Oak Ridge National Laboratory (ORNL) has been supporting the Defense Logistics Agency-Defense National Stockpile Center with stewardship of a thorium nitrate (ThN) stockpile. The effort for fiscal year 2002 was to prepare a sampling and analysis plan and to use the activities developed in the plan to characterize the ThN stockpile. The sampling was performed in June and July 2002 by RWE NUKEM with oversight by ORNL personnel. The analysis was performed by Southwest Research Institute of San Antonio, Texas, and data validation was performed by NFT, Inc., of Oak Ridge, Tennessee. Of the {approx} 21,000 drums in the stockpile, 99 were sampled and 53 were analyzed for total metals composition, radiological constituents (using alpha and gamma spectrometry), and oxidizing characteristics. Each lot at the Curtis Bay Depot was sampled. Several of the samples were also analyzed for density. The average density of the domestic ThN was found to be 1.89 {+-} 0.08 g/cm{sup 3}. The oxidizer test was performed following procedures issued by the United Nations in 1999. Test results indicated that none of the samples tested was a Division 5.1 oxidizer per Department of Transportation definition. The samples were analyzed for total metals following the U.S. Environmental Protection Agency methods SW-846-6010B and 6020 (EPA 2003) using a combination of inductively coupled plasma--atomic emission spectroscopy and inductively coupled plasma--mass spectroscopy techniques. The results were used to compare the composition of the eight Resource Conservation and Recovery Act metals present in the sample (arsenic, barium, cadmium, chromium, lead, mercury, selenium, and silver) to regulatory limits. None of the samples was found to be hazardous for toxicity characteristics. The radiological analyses confirmed, when possible, the results obtained by the inductively coupled plasma analyses. These results--combined with the historical process knowledge acquired on the material

  3. Functional roles of CymA and NapC in reduction of nitrate and nitrite by Shewanella putrefaciens W3-18-1

    Energy Technology Data Exchange (ETDEWEB)

    Beliav, Alex; Qiu, Dongru; Fredrickson, James K.; Wei, Hehong; Nealson, Kenneth H.; Xia, Ming; Zhou, Jizhong; Dai, Jingcheng; Shi, Liang; Tiedje, James M.; Romine, Margaret F.

    2016-06-01

    Shewanella putrefaciens W3-18-1 harbours two periplasmic nitrate reductase (Nap) gene clusters, NapC-associated nap-alpha (napEDABC) and CymA-dependent nap-beta (napDAGHB), for dissimilatory nitrate respiration. CymA is a member of the NapC/NirT quinol dehydrogenase family and acts as a hub to support different respiratory pathways, including those on iron [Fe(III)] and manganese [Mn(III, IV)] (hydr)oxide, nitrate, nitrite, fumarate and arsenate in Shewanella strains. However, in our analysis it was shown that another NapC/NirT family protein, NapC, was only involved in nitrate reduction, although both CymA and NapC can transfer quinol-derived electrons to a periplasmic terminal reductase or an electron acceptor. Furthermore, our results showed that NapC could only interact specifically with the Nap-alpha nitrate reductase while CymA could interact promiscuously with Nap-alpha, Nap-beta and the NrfA nitrite reductase for nitrate and nitrite reduction. To further explore the difference in specificity, site-directed mutagenesis on both CymA and NapC was conducted and the phenotypic changes in nitrate and nitrite reduction were tested. Our analyses demonstrated that the Lys-91 residue played a key role in nitrate reduction for quinol oxidation and the Asp-166 residue might influence the maturation of CymA. The Asp-97 residue might be one of the key factors that influence the interaction of CymA with the cytochromes NapB and NrfA.

  4. Functional roles of CymA and NapC in reduction of nitrate and nitrite by Shewanella putrefaciens W3-18-1.

    Science.gov (United States)

    Wei, Hehong; Dai, Jingcheng; Xia, Ming; Romine, Margaret F; Shi, Liang; Beliav, Alex; Tiedje, James M; Nealson, Kenneth H; Fredrickson, James K; Zhou, Jizhong; Qiu, Dongru

    2016-06-01

    Shewanella putrefaciens W3-18-1 harbours two periplasmic nitrate reductase (Nap) gene clusters, NapC-associated nap-alpha (napEDABC) and CymA-dependent nap-beta (napDAGHB), for dissimilatory nitrate respiration. CymA is a member of the NapC/NirT quinol dehydrogenase family and acts as a hub to support different respiratory pathways, including those on iron [Fe(III)] and manganese [Mn(III, IV)] (hydr)oxide, nitrate, nitrite, fumarate and arsenate in Shewanella strains. However, in our analysis it was shown that another NapC/NirT family protein, NapC, was only involved in nitrate reduction, although both CymA and NapC can transfer quinol-derived electrons to a periplasmic terminal reductase or an electron acceptor. Furthermore, our results showed that NapC could only interact specifically with the Nap-alpha nitrate reductase while CymA could interact promiscuously with Nap-alpha, Nap-beta and the NrfA nitrite reductase for nitrate and nitrite reduction. To further explore the difference in specificity, site-directed mutagenesis on both CymA and NapC was conducted and the phenotypic changes in nitrate and nitrite reduction were tested. Our analyses demonstrated that the Lys-91 residue played a key role in nitrate reduction for quinol oxidation and the Asp-166 residue might influence the maturation of CymA. The Asp-97 residue might be one of the key factors that influence the interaction of CymA with the cytochromes NapB and NrfA.

  5. 77 FR 65532 - Solid Fertilizer Grade Ammonium Nitrate From the Russian Federation: Notice of Rescission of...

    Science.gov (United States)

    2012-10-29

    ... International Trade Administration Solid Fertilizer Grade Ammonium Nitrate From the Russian Federation: Notice... the antidumping duty order on solid fertilizer grade ammonium nitrate (ammonium nitrate) from the... Administrative Review: Solid Fertilizer Grade Ammonium Nitrate (Ammonium Nitrate) from the Russian...

  6. New ALPHA-2 magnet

    CERN Multimedia

    Anaïs Schaeffer

    2012-01-01

    On 21 June, members of the ALPHA collaboration celebrated the handover of the first solenoid designed for the ALPHA-2 experiment. The magnet has since been successfully installed and is working well.   Khalid Mansoor, Sumera Yamin and Jeffrey Hangst in front of the new ALPHA-2 solenoid. “This was the first of three identical solenoids that will be installed between now and September, as the rest of the ALPHA-2 device is installed and commissioned,” explains ALPHA spokesperson Jeffrey Hangst. “These magnets are designed to allow us to transfer particles - antiprotons, electrons and positrons - between various parts of the new ALPHA-2 device by controlling the transverse size of the particle bunch that is being transferred.” Sumera Yamin and Khalid Mansoor, two Pakistani scientists from the National Centre for Physics in Islamabad, came to CERN in February specifically to design and manufacture these magnets. “We had the chance to work on act...

  7. Lyman Alpha Control

    CERN Document Server

    Nielsen, Daniel Stefaniak

    2015-01-01

    This document gives an overview of how to operate the Lyman Alpha Control application written in LabVIEW along with things to watch out for. Overview of the LabVIEW code itself as well as the physical wiring of and connections from/to the NI PCI-6229 DAQ box is also included. The Lyman Alpha Control application is the interface between the ALPHA sequencer and the HighFinesse Wavelength Meter as well as the Lyman Alpha laser setup. The application measures the wavelength of the output light from the Lyman Alpha cavity through the Wavelength Meter. The application can use the Wavelength Meter’s PID capabilities to stabilize the Lyman Alpha laser output as well as switch between up to three frequencies.

  8. Denitrification of nitrate waste solutions

    Energy Technology Data Exchange (ETDEWEB)

    Bertolami, R.J.; Chao, E.I.; Choi, W.M.; Johnson, B.R.; Varlet, J.L.P.

    1976-04-26

    Growth rates for the denitrifying bacteria Pseudomonas Stutzeri were studied to minimize the time necessary to start up a bacterial denitrification reactor. Batch experiments were performed in nine 250-ml Erlenmeyer flasks, a 7-liter fermentor, and a 67-liter fermentor. All reactors maintained an anaerobic environment. Initial microorganism inoculum concentration was varied over four orders of magnitude. Initial nitrate and substrate carbon concentrations were varied from 200 to 6000 ppm and from 56 to 1596 ppm, respectively, with a carbon-to-nitrogen weight ratio of 1.18. In all experiments, except those with the highest initial substrate-to-bacteria ratio, no growth was observed due to substrate depletion during the lag period. In those experiments which did exhibit an increase in bacterial population, growth also stopped due to substrate depletion. A model simulating microbe growth during the induction period was developed, but insufficient data were available to properly adjust the model constants. Because of this, the model does not accurately predict microbe growth. The metabolism of Pseudomonas Stutzeri was studied in detail. This resulted in a prediction of the denitrification stoichiometry during steady state reactor operation. Iron was found to be an important component for bacterial anabolism.

  9. Application of nitrate to enhance biodegradation of gasoline components in soil by indigenous microorganisms under anoxic condition.

    Science.gov (United States)

    Yang, Su-Cai; Song, Yun; Wang, Dong; Wei, Wen-Xia; Yang, Yan; Men, Bin; Li, Jia-Bin

    2016-01-01

    Anaerobic/anoxic biodegradation of hydrocarbons offers an attractive approach to the removal of these compounds from polluted environments such as aquifers, aquatic sediments, submerged soils and subsurface soils. The application of nitrate was investigated to accelerate the degradation of gasoline components such as mono-aromatic hydrocarbons and total petroleum hydrocarbons (TPH) in soil by indigenous microorganisms under anoxic condition. The addition of nitrate had little effect on the degradation of mono-aromatic hydrocarbons m- & p-xylene, o-xylene, sec-butylbenzene and 1,2,4-trimethylbenzene, but facilitated the degradation of TPH (C6-C12) and mono-aromatic hydrocarbons toluene and ethylbenzene markedly. Furthermore, the more nitrate added, the higher the percentage of toluene, ethylbenzene and TPH (C6-C12) degraded after 180 days of anoxic incubation. Microorganisms capable of degrading toluene, ethylbenzene and TPH (C6-C12) with nitrate as the electron acceptor under anaerobic/anoxic condition are composed predominantly of Alpha-, Beta-, Gamma- or Delta-proteobacteria. Beta- and Gamma-proteobacteria were the main components of indigenous microorganisms, and accounted for 83-100% of the total amount of indigenous microorganisms in soil used in this study. Furthermore, the total amount of indigenous microorganisms increased with nitrate added. The addition of nitrate stimulated the growth of indigenous microorganisms, and therefore facilitated the degradation of toluene, ethylbenzene and TPH (C6-C12).

  10. Removal of Nitrate from Groundwater by Cyanobacteria: Quantitative Assessment of Factors Influencing Nitrate Uptake

    OpenAIRE

    Hu, Qiang; Westerhoff, Paul; Vermaas, Wim

    2000-01-01

    The feasibility of biologically removing nitrate from groundwater was tested by using cyanobacterial cultures in batch mode under laboratory conditions. Results demonstrated that nitrate-contaminated groundwater, when supplemented with phosphate and some trace elements, can be used as growth medium supporting vigorous growth of several strains of cyanobacteria. As cyanobacteria grew, nitrate was removed from the water. Of three species tested, Synechococcus sp. strain PCC 7942 displayed the h...

  11. ROE Wet Nitrate Deposition 1989-1991

    Data.gov (United States)

    U.S. Environmental Protection Agency — The raster data represent the amount of wet nitrate deposition in kilograms per hectare from 1989 to 1991. Summary data in this indicator were provided by EPA’s...

  12. ROE Wet Nitrate Deposition 2011-2013

    Data.gov (United States)

    U.S. Environmental Protection Agency — The raster data represent the amount of wet nitrate deposition in kilograms per hectare from 2011 to 2013. Summary data in this indicator were provided by EPA’s...

  13. Spectrophotometric Determination of Nitrate and Phosphate Levels ...

    African Journals Online (AJOL)

    MBI

    2013-04-09

    Apr 9, 2013 ... Drinking Water Samples in The Vicinity of Irrigated Farmlands of Kura ... of Kura irrigated farmlands using polythene plastic containers and were analysed for the nitrate and ... polythene bottles, the bottles were covered with.

  14. Nitrate reduction in geologically heterogeneous catchments

    DEFF Research Database (Denmark)

    Refsgaard, Jens Christian; Auken, Esben; Bamberg, C.A.

    2014-01-01

    In order to fulfil the requirements of the EU Water Framework Directive nitrate load from agricultural areas to surface water in Denmark needs to be reduced by about 40%. The regulations imposed until now have been uniform, i.e. the same restrictions for all areas independent of the subsurface...... conditions. Studies have shown that on a national basis about 2/3 of the nitrate leaching from the root zone is reduced naturally, through denitrification, in the subsurface before reaching the streams. Therefore, it is more cost-effective to identify robust areas, where nitrate leaching through the root...... the entire catchment. However, as distributed models often do not include local scale hydrogeological heterogeneities, they are typically not able to make accurate predictions at scales smaller than they are calibrated. We present a framework for assessing nitrate reduction in the subsurface...

  15. Nitrate Waste Treatment Sampling and Analysis Plan

    Energy Technology Data Exchange (ETDEWEB)

    Vigil-Holterman, Luciana R. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Martinez, Patrick Thomas [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Garcia, Terrence Kerwin [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-07-05

    This plan is designed to outline the collection and analysis of nitrate salt-bearing waste samples required by the New Mexico Environment Department- Hazardous Waste Bureau in the Los Alamos National Laboratory (LANL) Hazardous Waste Facility Permit (Permit).

  16. Qualitative Determination of Nitrate with Triphenylbenzylphosphonium Chloride.

    Science.gov (United States)

    Berry, Donna A.; Cole, Jerry J.

    1984-01-01

    Discusses two procedures for the identification of nitrate, the standard test ("Brown Ring" test) and a new procedure using triphenylbenzylphosphonium chloride (TPBPC). Effectiveness of both procedures is compared, with the TPBPC test proving to be more sensitive and accurate. (JM)

  17. 76 FR 70366 - Ammonium Nitrate Security Program

    Science.gov (United States)

    2011-11-14

    ...- accessible Internet access could obtain the access necessary to register online. 3. How to best notify... ammonium nitrate sellers (AN Sellers) when it is not possible for an AN Seller to verify the identity of...

  18. 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....

  19. Interpreting EEG alpha activity.

    Science.gov (United States)

    Bazanova, O M; Vernon, D

    2014-07-01

    Exploring EEG alpha oscillations has generated considerable interest, in particular with regards to the role they play in cognitive, psychomotor, psycho-emotional and physiological aspects of human life. However, there is no clearly agreed upon definition of what constitutes 'alpha activity' or which of the many indices should be used to characterize it. To address these issues this review attempts to delineate EEG alpha-activity, its physical, molecular and morphological nature, and examine the following indices: (1) the individual alpha peak frequency; (2) activation magnitude, as measured by alpha amplitude suppression across the individual alpha bandwidth in response to eyes opening, and (3) alpha "auto-rhythmicity" indices: which include intra-spindle amplitude variability, spindle length and steepness. Throughout, the article offers a number of suggestions regarding the mechanism(s) of alpha activity related to inter and intra-individual variability. In addition, it provides some insights into the various psychophysiological indices of alpha activity and highlights their role in optimal functioning and behavior.

  20. The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds.

    Science.gov (United States)

    Chopin, Franck; Orsel, Mathilde; Dorbe, Marie-France; Chardon, Fabien; Truong, Hoai-Nam; Miller, Anthony J; Krapp, Anne; Daniel-Vedele, Françoise

    2007-05-01

    In higher plants, nitrate is taken up by root cells where Arabidopsis thaliana NITRATE TRANSPORTER2.1 (ATNRT2.1) chiefly acts as the high-affinity nitrate uptake system. Nitrate taken up by the roots can then be translocated from the root to the leaves and the seeds. In this work, the function of the ATNRT2.7 gene, one of the seven members of the NRT2 family in Arabidopsis, was investigated. High expression of the gene was detected in reproductive organs and peaked in dry seeds. beta-Glucuronidase or green fluorescent protein reporter gene expression driven by the ATNRT2.7 promoter confirmed this organ specificity. We assessed the capacity of ATNRT2.7 to transport nitrate in Xenopus laevis oocytes or when it is expressed ectopically in mutant plants deficient in nitrate transport. We measured the impact of an ATNRT2.7 mutation and found no difference from the wild type during vegetative development. By contrast, seed nitrate content was affected by overexpression of ATNRT2.7 or a mutation in the gene. Finally, we showed that this nitrate transporter protein was localized to the vacuolar membrane. Our results demonstrate that ATNRT2.7 plays a specific role in nitrate accumulation in the seed.