Sample records for axonal transport phosphorylation

  1. Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of neurofilaments.

    LENUS (Irish Health Repository)

    Yates, Darran M


    Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.

  2. Functional Impact of Corticotropin-Releasing Factor Exposure on Tau Phosphorylation and Axon Transport.

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    Michelle H Le

    Full Text Available Stress exposure or increased levels of corticotropin-releasing factor (CRF induce hippocampal tau phosphorylation (tau-P in rodent models, a process that is dependent on the type-1 CRF receptor (CRFR1. Although these preclinical studies on stress-induced tau-P provide mechanistic insight for epidemiological work that identifies stress as a risk factor for Alzheimer's disease (AD, the actual impact of stress-induced tau-P on neuronal function remains unclear. To determine the functional consequences of stress-induced tau-P, we developed a novel mouse neuronal cell culture system to explore the impact of acute (0.5hr and chronic (2hr CRF treatment on tau-P and integral cell processes such as axon transport. Consistent with in vivo reports, we found that chronic CRF treatment increased tau-P levels and caused globular accumulations of phosphorylated tau in dendritic and axonal processes. Furthermore, while both acute and chronic CRF treatment led to significant reduction in CREB activation and axon transport of brain-derived neurotrophic factor (BDNF, this was not the case with mitochondrial transport. Acute CRF treatment caused increased mitochondrial velocity and distance traveled in neurons, while chronic CRF treatment modestly decreased mitochondrial velocity and greatly increased distance traveled. These results suggest that transport of cellular energetics may take priority over growth factors during stress. Tau-P was required for these changes, as co-treatment of CRF with a GSK kinase inhibitor prevented CRF-induced tau-P and all axon transport changes. Collectively, our results provide mechanistic insight into the consequences of stress peptide-induced tau-P and provide an explanation for how chronic stress via CRF may lead to neuronal vulnerability in AD.

  3. Age-related changes in axonal transport. (United States)

    Frolkis, V V; Tanin, S A; Gorban, Y N


    In rats the rate of axonal transport (AT) or radiolabeled material decreased in the ventral roots of the spinal cord and the vagal and hypoglossal nerves with aging. A maximum AT deceleration in old age was observed in the vagus. The uncoupling of oxidative phosphorylation, inhibition of glycolysis and hypoxia induced a greater AT deceleration in old rats as compared to adults. Small doses of sodium fluoride accelerated AT, and this correlated with a rise in cAMP levels in ventral roots. High doses of sodium fluoride decelerated AT more markedly in old rats. It was shown that anabolic hormones (sex steroids and thyroxine) accelerated AT in both adult and old rats, whereas insulin induced a rise in AT rate in only adults. The catabolic steroid, hydrocortisone decelerated AT. In old rats castration diminished AT, while thyroidectomy had no effect. It was also shown that hydrocortisone and testosterone were transported along axons, reached fibers of the skeletal muscles, and hyperpolarized the plasma membrane. In old age the latent period was extended. Following 73 to 74 days of irradiation, AT slowed down in all the nerves studied in both adult and old rats. Following irradiation hormonal effects on AT changed, for example, the stimulatory effect of estradiol became weak, especially in old rats. Changes in AT could be an important mechanism of disordering the growth of neurons and innervated cells in old age.

  4. Cargo distributions differentiate pathological axonal transport impairments. (United States)

    Mitchell, Cassie S; Lee, Robert H


    Axonal transport is an essential process in neurons, analogous to shipping goods, by which energetic and cellular building supplies are carried downstream (anterogradely) and wastes are carried upstream (retrogradely) by molecular motors, which act as cargo porters. Impairments in axonal transport have been linked to devastating and often lethal neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis, Huntington's, and Alzheimer's. Axonal transport impairment types include a decrease in available motors for cargo transport (motor depletion), the presence of defective or non-functional motors (motor dilution), and the presence of increased or larger cargos (protein aggregation). An impediment to potential treatment identification has been the inability to determine what type(s) of axonal transport impairment candidates that could be present in a given disease. In this study, we utilize a computational model and common axonal transport experimental metrics to reveal the axonal transport impairment general characteristics or "signatures" that result from three general defect types of motor depletion, motor dilution, and protein aggregation. Our results not only provide a means to discern these general impairments types, they also reveal key dynamic and emergent features of axonal transport, which potentially underlie multiple impairment types. The identified characteristics, as well as the analytical method, can be used to help elucidate the axonal transport impairments observed in experimental and clinical data. For example, using the model-predicted defect signatures, we identify the defect candidates, which are most likely to be responsible for the axonal transport impairments in the G93A SOD1 mouse model of ALS. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Dynamics of mitochondrial transport in axons

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    Robert Francis Niescier


    Full Text Available The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons.

  6. Synaptic Democracy and Vesicular Transport in Axons (United States)

    Bressloff, Paul C.; Levien, Ethan


    Synaptic democracy concerns the general problem of how regions of an axon or dendrite far from the cell body (soma) of a neuron can play an effective role in neuronal function. For example, stimulated synapses far from the soma are unlikely to influence the firing of a neuron unless some sort of active dendritic processing occurs. Analogously, the motor-driven transport of newly synthesized proteins from the soma to presynaptic targets along the axon tends to favor the delivery of resources to proximal synapses. Both of these phenomena reflect fundamental limitations of transport processes based on a localized source. In this Letter, we show that a more democratic distribution of proteins along an axon can be achieved by making the transport process less efficient. This involves two components: bidirectional or "stop-and-go" motor transport (which can be modeled in terms of advection-diffusion), and reversible interactions between motor-cargo complexes and synaptic targets. Both of these features have recently been observed experimentally. Our model suggests that, just as in human societies, there needs to be a balance between "efficiency" and "equality".

  7. EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment

    Czech Academy of Sciences Publication Activity Database

    Eva, R.; Koseki, H.; Kanamarlapudi, V.; Fawcett, James


    Roč. 130, č. 21 (2017), s. 3663-3675 ISSN 0021-9533 Institutional support: RVO:68378041 Keywords : axon regeneration * axon transport * neuronal polarisation Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 4.431, year: 2016

  8. Kinematics of turnaround and retrograde axonal transport

    International Nuclear Information System (INIS)

    Snyder, R.E.


    Rapid axonal transport of a pulse of 35 S-methionine-labelled material was studied in vitro in the sensory neurons of amphibian sciatic nerve using a position-sensitive detector. For 10 nerves studied at 23.0 +/- 0.2 degrees C it was found that a pulse moved in the anterograde direction characterized by front edge, peak, and trailing edge transport rates of (mm/d) 180.8 +/- 2.2 (+/- SEM), 176.6 +/- 2.3, and 153.7 +/- 3.0, respectively. Following its arrival at a distal ligature, a smaller pulse was observed to move in the retrograde direction characterized by front edge and peak transport rates of 158.0 +/- 7.3 and 110.3 +/- 3.5, respectively, indicating that retrograde transport proceeds at a rate of 0.88 +/- 0.04 that of anterograde. The retrograde pulse was observed to disperse at a rate greater than the anterograde. Reversal of radiolabel at the distal ligature began 1.49 +/- 0.15 h following arrival of the first radiolabel. Considerable variation was seen between preparations in the way radiolabel accumulated in the end (ligature) regions of the nerve. Although a retrograde pulse was seen in all preparations, in 7 of 10 preparations there was no evidence of this pulse accumulating within less than 2-3 mm of a proximal ligature; however, accumulation was observed within less than 5 mm in all preparations

  9. Regulation of Cdh1-APC Function in Axon Growth by Cdh1 Phosphorylation


    Huynh, Mai Anh; Stegmüller, Judith; Litterman, Nadia; Bonni, Azad


    The ubiquitin ligase Cdh1-anaphase promoting complex (Cdh1-APC) plays a key role in the control of axonal morphogenesis in the mammalian brain, but the mechanisms that regulate neuronal Cdh1-APC function remain incompletely understood. Here, we have characterized the effect of phosphorylation of Cdh1 at cyclin-dependent kinase (Cdk) sites on Cdh1-APC function in neurons. We replaced nine conserved sites of Cdk-induced Cdh1 phosphorylation with alanine (9A) or aspartate (9D) to mimic hypo- or ...

  10. Reduced axonal transport in Parkinson's disease cybrid neurites is restored by light therapy

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    De Taboada Luis


    Full Text Available Abstract Background It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson's disease (PD. Mitochondria supply the adenosine triphosphate (ATP needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid "cytoplasmic hybrid" neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT. The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC enhances mitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT. Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser (50 mW/cm2 for 40 seconds. Oxygen utilization and assembly of mtETC complexes were also determined. Results The velocity of mitochondrial movement in PD cybrid neuronal cells (0.175 +/- 0.005 SEM was significantly reduced (p Conclusion The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatment to improve neuronal function in patients with PD.

  11. Riluzole protects against glutamate-induced slowing of neurofilament axonal transport.

    LENUS (Irish Health Repository)

    Stevenson, Alison


    Riluzole is the only drug approved for the treatment of amyotrophic lateral sclerosis (ALS) but its precise mode of action is not properly understood. Damage to axonal transport of neurofilaments is believed to be part of the pathogenic mechanism in ALS and this has been linked to defective glutamate handling and increased phosphorylation of neurofilament side-arm domains. Here, we show that riluzole protects against glutamate-induced slowing of neurofilament transport. Protection is associated with decreased neurofilament side-arm phosphorylation and inhibition of the activities of two neurofilament kinases, ERK and p38 that are activated in ALS. Thus, the anti-glutamatergic properties of riluzole include protection against glutamate-induced changes to neurofilament phosphorylation and transport.

  12. Mechanistic logic underlying the axonal transport of cytosolic proteins (United States)

    Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit


    Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

  13. Loss of fractalkine signaling exacerbates axon transport dysfunction in a chronic model of glaucoma

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    Kevin T Breen


    Full Text Available Neurodegeneration in glaucoma results in decline and loss of retinal ganglion cells (RGCs, and is associated with activation of myeloid cells such as microglia and macrophages. The chemokine fractalkine (FKN or Cx3cl1 mediates communication from neurons to myeloid cells. Signaling through its receptor Cx3cr1 has been implicated in multiple neurodegenerative diseases, but the effects on neuronal pathology are variable. Since it is unknown how FKN-mediated crosstalk influences RGC degeneration in glaucoma, we assessed this in a chronic mouse model, DBA/2J. We analyzed a DBA/2J substrain deficient in Cx3cr1, and compared compartmentalized RGC degeneration and myeloid cell responses to those in standard DBA/2J mice. We found that loss of FKN signaling exacerbates axon transport dysfunction, an early event in neurodegeneration, with a significant increase in RGCs with somal accumulation of the axonal protein phosphorylated neurofilament, and reduced retinal expression of genes involved in axon transport, Kif1b and Atp8a2. There was no change in the loss of Brn3-positive RGCs, and no difference in the extent of damage to the proximal optic nerve, suggesting that the loss of fractalkine signaling primarily affects axon transport. Since Cx3cr1 is specifically expressed in myeloid cells, we assessed changes in retinal microglial number and activation, changes in gene expression, and the extent of macrophage infiltration. We found that loss of fractalkine signaling led to innate immune changes within the retina, including increased infiltration of peripheral macrophages and upregulated nitric oxide synthase-2 (Nos-2 expression in myeloid cells, which contributes to the production of NO and can promote axon transport deficits. In contrast, resident retinal microglia appeared unchanged either in number, morphology, or expression of the myeloid activation marker ionized calcium binding adaptor molecule 1 (Iba1. There was also no significant increase in the

  14. Axonal Actin Transport Driven By Metastable Actin Filaments (United States)

    Chakrabarty, Nilaj; Ganguly, Archan; Roy, Subhojit; Jung, Peter

    Actin is one of the key constituents of the neuronal cytoskeleton and is responsible for driving important cellular processes like axon elongation. Axonal actin is synthesized in the cell body and transported at rates of 0.25 - 3 mm/day, as shown by in-vivo pulse-chase radiolabelling studies. However, the underlying transport mechanisms are unknown. Recent experiments in cultured neurons have revealed a dynamic network of metastable actin filaments (actin trails). Actin trails seem to originate from focal actin hotspots which colocalize with stationary endosomes. Interestingly, the number of actin trails extending anterogradely is higher than the ones extending retrogradely. We hypothesize that the bulk axonal transport of actin originates from this directional asymmetry of the number of actin trails. To test this, we constructed a computational model of actin trail growth and simulated the pulse-chase experiment. In our model, local, metastable trails, which grow with their barbed ends anchored to the hotspots, drive the bulk anterograde transport. Our results indicate that the observed bias of the nucleation probabilities and the elongation rate of actin trails are sufficient to drive the bulk transport of actin at rates that agree with in-vivo pulse chase experiments.

  15. Internalization and Axonal Transport of the HIV Glycoprotein gp120 (United States)

    Berth, Sarah; Caicedo, Hector Hugo; Sarma, Tulika; Morfini, Gerardo


    The HIV glycoprotein gp120, a neurotoxic HIV glycoprotein that is overproduced and shed by HIV-infected macrophages, is associated with neurological complications of HIV such as distal sensory polyneuropathy, but interactions of gp120 in the peripheral nervous system remain to be characterized. Here, we demonstrate internalization of extracellular gp120 in a manner partially independent of binding to its coreceptor CXCR4 by F11 neuroblastoma cells and cultured dorsal root ganglion neurons. Immunocytochemical and pharmacological experiments indicate that gp120 does not undergo trafficking through the endolysosomal pathway. Instead, gp120 is mainly internalized through lipid rafts in a cholesterol-dependent manner, with a minor fraction being internalized by fluid phase pinocytosis. Experiments using compartmentalized microfluidic chambers further indicate that, after internalization, endocytosed gp120 selectively undergoes retrograde but not anterograde axonal transport from axons to neuronal cell bodies. Collectively, these studies illuminate mechanisms of gp120 internalization and axonal transport in peripheral nervous system neurons, providing a novel framework for mechanisms for gp120 neurotoxicity. PMID:25636314

  16. Selective rab11 transport and the intrinsic regenerative ability of CNS axons. (United States)

    Koseki, Hiroaki; Donegá, Matteo; Lam, Brian Yh; Petrova, Veselina; van Erp, Susan; Yeo, Giles Sh; Kwok, Jessica Cf; Ffrench-Constant, Charles; Eva, Richard; Fawcett, James W


    Neurons lose intrinsic axon regenerative ability with maturation, but the mechanism remains unclear. Using an in-vitro laser axotomy model, we show a progressive decline in the ability of cut CNS axons to form a new growth cone and then elongate. Failure of regeneration was associated with increased retraction after axotomy. Transportation into axons becomes selective with maturation; we hypothesized that selective exclusion of molecules needed for growth may contribute to regeneration decline. With neuronal maturity rab11 vesicles (which carry many molecules involved in axon growth) became selectively targeted to the somatodendritic compartment and excluded from axons by predominant retrograde transport However, on overexpression rab11 was mistrafficked into proximal axons, and these axons showed less retraction and enhanced regeneration after axotomy. These results suggest that the decline of intrinsic axon regenerative ability is associated with selective exclusion of key molecules, and that manipulation of transport can enhance regeneration.

  17. Retinal ganglion cells: Energetics, compartmentation, axonal transport, cytoskeletons and vulnerability. (United States)

    Yu, Dao-Yi; Cringle, Stephen J; Balaratnasingam, Chandrakumar; Morgan, William H; Yu, Paula K; Su, Er-Ning


    Retinal ganglion cells (RGCs) are specialized projection neurons that relay an immense amount of visual information from the retina to the brain. RGC signal inputs are collected by dendrites and output is distributed from the cell body via very thin (0.5-1 μm) and long (∼50 mm) axons. The RGC cell body is larger than other retinal neurons, but is still only a very small fraction (one ten thousandths) of the length and total surface area of the axon. The total distance traversed by RGCs extends from the retina, starting from synapses with bipolar and amacrine cells, to the brain, to synapses with neurons in the lateral geniculate nucleus. This review will focus on the energy demands of RGCs and the relevant tissues that surround them. RGC survival and function unexceptionally depends upon free energy, predominantly adenosine triphosphate (ATP). RGC energy metabolism is vastly different when compared to that of the photoreceptors. Each subcellular component of the RGC is remarkably different in terms of structure, function and extracellular environment. The energy demands and distribution of each component are also distinct as evidenced by the uneven distribution of mitochondria and ATP within the RGC - signifying the presence of intracellular energy gradients. In this review we will describe RGCs as having four subcellular components, (1) Dendrites, (2) Cell body, (3) Non-myelinated axon, including intraocular and optic nerve head portions, and (4) Myelinated axon, including the intra-orbital and intracranial portions. We will also describe how RGCs integrate information from each subcellular component in order achieve intracellular homeostatic stability as well as respond to perturbations in the extracellular environment. The possible cellular mechanisms such as axonal transport and axonal cytoskeleton proteins that are involved in maintaining RGC energy homeostasis during normal and disease conditions will also be discussed in depth. The emphasis of this

  18. Efficient retrograde transport of pseudorabies virus within neurons requires local protein synthesis in axons. (United States)

    Koyuncu, Orkide O; Perlman, David H; Enquist, Lynn W


    After replicating in epithelial cells, alphaherpesviruses such as pseudorabies virus (PRV) invade axons of peripheral nervous system neurons and undergo retrograde transport toward the distant cell bodies. Although several viral proteins engage molecular motors to facilitate transport, the initial steps and neuronal responses to infection are poorly understood. Using compartmented neuron cultures to physically separate axon infection from cell bodies, we found that PRV infection induces local protein synthesis in axons, including proteins involved in cytoskeletal remodeling, intracellular trafficking, signaling, and metabolism. This rapid translation of axonal mRNAs is required for efficient PRV retrograde transport and infection of cell bodies. Furthermore, induction of axonal damage, which also induces local protein synthesis, prior to infection reduces virion trafficking, suggesting that host damage signals and virus particles compete for retrograde transport. Thus, similar to axonal damage, virus infection induces local protein translation in axons, and viruses likely exploit this response for invasion. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Axonal transport and axon sprouting in the adult rat dentate gyrus: an autoradiographic study

    International Nuclear Information System (INIS)

    Goldowitz, D.; Cotman, C.W.


    In response to an entorhinal lesion, the commissural and associational afferents to the dentate gyrus have been shown to expand beyond their normal terminal zone into the area denervated by the entorhinal lesion. The present study has investigated the axonal transport of [ 3 H]-labeled proteins in the commissural and associational projections following an entorhinal lesion. Injections of [ 3 H]proline, [ 3 H]leucine or [ 3 H)fucose were given in the vicinity of the commissural and associational cells of origin before, immediately subsequent to, or at 5 to 15 days after the entorhinal lesion. The disposition of previously- or newly-synthesized proteins was examined in the commissural and associational terminal field at different times after an entorhinal lesion by light-microscopic autoradiography. (author)

  20. Neurogenetics of slow axonal transport: from cells to animals. (United States)

    Sadananda, Aparna; Ray, Krishanu


    Slow axonal transport is a multivariate phenomenon implicated in several neurodegenerative disorders. Recent reports have unraveled the molecular basis of the transport of certain slow component proteins, such as the neurofilament subunits, tubulin, and certain soluble enzymes such as Ca(2+)/calmodulin-dependent protein kinase IIa (CaM kinase IIa), etc., in tissue cultured neurons. In addition, genetic analyses also implicate microtubule-dependent motors and other housekeeping proteins in this process. However, the biological relevance of this phenomenon is not so well understood. Here, the authors have discussed the possibility of adopting neurogenetic analyses in multiple model organisms to correlate molecular level measurements of the slow transport phenomenon to animal behavior, thus facilitating the investigation of its biological efficacy.

  1. Effect of vesicle traps on traffic jam formation in fast axonal transport. (United States)

    Kuznetsov, A V


    The purpose of this paper is to develop a model for simulation of the formation of organelle traps in fast axonal transport. Such traps may form in the regions of microtubule polar mismatching. Depending on the orientation of microtubules pointing toward the trap region, these traps can accumulate either plus-end or minus-end oriented vesicles. The model predicts that the maximum concentrations of organelles occur at the boundaries of the trap regions; the overall concentration of organelles in the axon with traps is greatly increased compared to that in a healthy axon, which is expected to contribute to mechanical damages of the axon. The organelle traps induce hindrance to organelle transport down the axon; the total organelle flux down the axon with traps is found to be significantly reduced compared to that in a healthy axon. Copyright 2010 Elsevier Inc. All rights reserved.

  2. Dynein is the motor for retrograde axonal transport of organelles

    International Nuclear Information System (INIS)

    Schnapp, B.J.; Reese, T.S.


    Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport

  3. The time course of ongoing activity during neuritis and following axonal transport disruption. (United States)

    Satkeviciute, Ieva; Goodwin, George; Bove, Geoffrey M; Dilley, Andrew


    Local nerve inflammation (neuritis) leads to ongoing activity and axonal mechanical sensitivity (AMS) along intact nociceptor axons, and disrupts axonal transport. This phenomenon forms the most feasible cause of radiating pain, such as sciatica. We have previously shown that axonal transport disruption without inflammation or degeneration also leads to AMS, but does not cause ongoing activity at the time point when AMS occurs, despite causing cutaneous hypersensitivity. However, there have been no systematic studies of ongoing activity during neuritis or non-inflammatory axonal transport disruption. In this study, we present the time course of ongoing activity from primary sensory neurons following neuritis and vinblastine-induced axonal transport disruption. Whereas 24% of C/slow Aδ-fiber neurons had ongoing activity during neuritis, few (disruption of axonal transport without inflammation does not lead to ongoing activity in sensory neurons, including nociceptors, but does cause a rapid and transient development of AMS. Since it is proposed that AMS underlies mechanically-induced radiating pain, and a transient disruption of axonal transport (as previously reported) leads to transient AMS, it follows that processes that disrupt axonal transport, such as neuritis, must persist to maintain AMS and the associated symptoms.

  4. Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid. (United States)

    Tsai, Shang-Yi A; Pokrass, Michael J; Klauer, Neal R; Nohara, Hiroshi; Su, Tsung-Ping


    Dysregulation of cyclin-dependent kinase 5 (cdk5) per relative concentrations of its activators p35 and p25 is implicated in neurodegenerative diseases. P35 has a short t½ and undergoes rapid proteasomal degradation in its membrane-bound myristoylated form. P35 is converted by calpain to p25, which, along with an extended t½, promotes aberrant activation of cdk5 and causes abnormal hyperphosphorylation of tau, thus leading to the formation of neurofibrillary tangles. The sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperone that is implicated in neuronal survival. However, the specific role of the Sig-1R in neurodegeneration is unclear. Here we found that Sig-1Rs regulate proper tau phosphorylation and axon extension by promoting p35 turnover through the receptor's interaction with myristic acid. In Sig-1R-KO neurons, a greater accumulation of p35 is seen, which results from neither elevated transcription of p35 nor disrupted calpain activity, but rather to the slower degradation of p35. In contrast, Sig-1R overexpression causes a decrease of p35. Sig-1R-KO neurons exhibit shorter axons with lower densities. Myristic acid is found here to bind Sig-1R as an agonist that causes the dissociation of Sig-1R from its cognate partner binding immunoglobulin protein. Remarkably, treatment of Sig-1R-KO neurons with exogenous myristic acid mitigates p35 accumulation, diminishes tau phosphorylation, and restores axon elongation. Our results define the involvement of Sig-1Rs in neurodegeneration and provide a mechanistic explanation that Sig-1Rs help maintain proper tau phosphorylation by potentially carrying and providing myristic acid to p35 for enhanced p35 degradation to circumvent the formation of overreactive cdk5/p25.

  5. The disruption of mitochondrial axonal transport is an early event in neuroinflammation

    DEFF Research Database (Denmark)

    Errea, Oihana; Moreno, Beatriz; Gonzalez-Franquesa, Alba


    of neuroprotective therapies. Energy depletion due to mitochondrial dysfunction has been postulated as an important step in the damage of axons. This prompted us to study the effects of acute inflammation and oxidative stress on the morphology, transport, and function of mitochondria in axons. METHODS: Mouse......BACKGROUND: In brain inflammatory diseases, axonal damage is one of the most critical steps in the cascade that leads to permanent disability. Thus, identifying the initial events triggered by inflammation or oxidative stress that provoke axonal damage is critical for the development...... in axons, increasing the proportion of stationary mitochondria in axons after LPS challenge. Indeed, the two challenges used produced different effects: inflammation mostly reducing retrograde transport and oxidative stress slightly enhancing retrograde transportation. CONCLUSIONS: Neuroinflammation...

  6. Organophosphate-Related Alterations in Myelin and Axonal Transport in the Living Mammalian Brain (United States)


    510. Duncan JE, Goldstein LS. 2006. The Genetics of Axonal Transport and Axonal Transport Disorders PLoS Genet . 2(9): e124. 25 Duysen EG, Li...Gitajn L, Rea W, Yang Y, Stein EA.2007. Cocaine -induced brain activation detected by dynamic manganese-enhanced magnetic resonance imaging (MEMRI

  7. The effect of myelinating Schwann cells on axons. (United States)

    Martini, R


    Myelinating Schwann cells control the number of neurofilaments and elevate the phosphorylation state of neurofilaments in the axon, eventually leading to the typical large axon caliber. Conversely, absence of myelin leads to lower amounts of neurofilaments, reduced phosphorylation levels, and smaller axon diameters. In addition, myelinating Schwann cells mediate the spacing of Na(+) channel clusters during development of the node of Ranvier. When axons are associated with mutant Schwann cells in inherited neuropathies, their calibers are reduced and their neurofilaments are less phosphorylated and more closely spaced. Also, axonal transport is reduced and axons degenerate at the distal ends of long nerves. Myelin-associated glycoprotein may mediate some aspects of Schwann cell-axon communication, but much remains to be learned about the molecular bases of Schwann cell-axon communication. Copyright 2001 John Wiley & Sons, Inc.

  8. KIF5C S176 Phosphorylation Regulates Microtubule Binding and Transport Efficiency in Mammalian Neurons.

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    Artur ePadzik


    Full Text Available Increased phosphorylation of the KIF5 anterograde motor is associated with impaired axonal transport and neurodegeneration, but paradoxically also with normal transport, though the details are not fully defined. JNK phosphorylates KIF5C on S176 in the motor domain; a site that we show is phosphorylated in brain. Microtubule pelleting assays demonstrate that phosphomimetic KIF5C(1-560S176D associates weakly with microtubules compared to KIF5C(1-560WT. Consistent with this, 50% of KIF5C(1-560S176D shows diffuse movement in neurons. However the remaining 50% remains microtubule bound and displays decreased pausing and increased bidirectional movement. The same directionality switching is observed with KIF5C(1-560WT in the presence of an active JNK chimera, MKK7-JNK. Yet, in cargo trafficking assays where peroxisome cargo is bound, KIF5C(1-560S176D-GFP-FRB transports normally to microtubule plus ends. We also find that JNK increases the ATP hydrolysis of KIF5C in vitro. These data suggest that phosphorylation of KIF5C-S176 primes the motor to either disengage entirely from microtubule tracks as previously observed in response to stress, or to display improved efficiency. The final outcome may depend on cargo load and motor ensembles.

  9. A high mitochondrial transport rate characterizes CNS neurons with high axonal regeneration capacity.

    Directory of Open Access Journals (Sweden)

    Romain Cartoni

    Full Text Available Improving axonal transport in the injured and diseased central nervous system has been proposed as a promising strategy to improve neuronal repair. However, the contribution of each cargo to the repair mechanism is unknown. DRG neurons globally increase axonal transport during regeneration. Because the transport of specific cargos after axonal insult has not been examined systematically in a model of enhanced regenerative capacity, it is unknown whether the transport of all cargos would be modulated equally in injured central nervous system neurons. Here, using a microfluidic culture system we compared neurons co-deleted for PTEN and SOCS3, an established model of high axonal regeneration capacity, to control neurons. We measured the axonal transport of three cargos (mitochondria, synaptic vesicles and late endosomes in regenerating axons and found that the transport of mitochondria, but not the other cargos, was increased in PTEN/SOCS3 co-deleted axons relative to controls. The results reported here suggest a pivotal role for this organelle during axonal regeneration.

  10. Neurobiology of axonal transport defects in motor neuron diseases: Opportunities for translational research? (United States)

    De Vos, Kurt J; Hafezparast, Majid


    Intracellular trafficking of cargoes is an essential process to maintain the structure and function of all mammalian cell types, but especially of neurons because of their extreme axon/dendrite polarisation. Axonal transport mediates the movement of cargoes such as proteins, mRNA, lipids, membrane-bound vesicles and organelles that are mostly synthesised in the cell body and in doing so is responsible for their correct spatiotemporal distribution in the axon, for example at specialised sites such as nodes of Ranvier and synaptic terminals. In addition, axonal transport maintains the essential long-distance communication between the cell body and synaptic terminals that allows neurons to react to their surroundings via trafficking of for example signalling endosomes. Axonal transport defects are a common observation in a variety of neurodegenerative diseases, and mutations in components of the axonal transport machinery have unequivocally shown that impaired axonal transport can cause neurodegeneration (reviewed in El-Kadi et al., 2007, De Vos et al., 2008; Millecamps and Julien, 2013). Here we review our current understanding of axonal transport defects and the role they play in motor neuron diseases (MNDs) with a specific focus on the most common form of MND, amyotrophic lateral sclerosis (ALS). Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Myelin-associated proteins labelled by slow axonal transport

    International Nuclear Information System (INIS)

    Giorgi, P.P.; DuBois, H.


    This paper deals with the problem of protein metabolism and provides evidence that the neuronal contribution to myelin metabolism may be restricted to lipids only. On the other hand this line of research led to the partial characterization of a group of neuronal proteins probably involved in axo-glial interactions subserving the onset of myelination and the structural maintenance of the mature myelin sheath. Intraocular injection of radioactive amino acids allows the study of the anterograde transport of labelled proteins along retinofugal fibres which are well myelinated. Myelin extracted from the optic nerve and tract under these conditions also contains labelled proteins. Three hypotheses are available to explain this phenomenon. To offer an explanation for this phenomenon the work was planned as follows. a) Characterization of the spatio-temporal pattern of labelling of myelin, in order to define the experimental conditions (survival time and region of the optic pathway to be studied) necessary to obtain maximal labelling. b) Characterization (by gel electrophoresis) of the myelin-associated proteins which become labelled by axonal transport, in order to work on a consistent pattern of labelling. c) Investigation of the possible mechanism responsible for the labelling of myelin-associated proteins. (Auth.)

  12. Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

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    Y Cao; X Jin; E Levin; H Huang; Y Zong; W Hendrickson; J Javitch; K Rajashankar; M Zhou; et al.


    Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

  13. Impaired JIP3-dependent axonal lysosome transport promotes amyloid plaque pathology. (United States)

    Gowrishankar, Swetha; Wu, Yumei; Ferguson, Shawn M


    Lysosomes robustly accumulate within axonal swellings at Alzheimer's disease (AD) amyloid plaques. However, the underlying mechanisms and disease relevance of such lysosome accumulations are not well understood. Motivated by these problems, we identified JNK-interacting protein 3 (JIP3) as an important regulator of axonal lysosome transport and maturation. JIP3 knockout mouse neuron primary cultures accumulate lysosomes within focal axonal swellings that resemble the dystrophic axons at amyloid plaques. These swellings contain high levels of amyloid precursor protein processing enzymes (BACE1 and presenilin 2) and are accompanied by elevated Aβ peptide levels. The in vivo importance of the JIP3-dependent regulation of axonal lysosomes was revealed by the worsening of the amyloid plaque pathology arising from JIP3 haploinsufficiency in a mouse model of AD. These results establish the critical role of JIP3-dependent axonal lysosome transport in regulating amyloidogenic amyloid precursor protein processing and support a model wherein Aβ production is amplified by plaque-induced axonal lysosome transport defects. © 2017 Gowrishankar et al.

  14. Methodological advances in imaging intravital axonal transport [version 1; referees: 3 approved

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    James N. Sleigh


    Full Text Available Axonal transport is the active process whereby neurons transport cargoes such as organelles and proteins anterogradely from the cell body to the axon terminal and retrogradely in the opposite direction. Bi-directional transport in axons is absolutely essential for the functioning and survival of neurons and appears to be negatively impacted by both aging and diseases of the nervous system, such as Alzheimer’s disease and amyotrophic lateral sclerosis. The movement of individual cargoes along axons has been studied in vitro in live neurons and tissue explants for a number of years; however, it is currently unclear as to whether these systems faithfully and consistently replicate the in vivo situation. A number of intravital techniques originally developed for studying diverse biological events have recently been adapted to monitor axonal transport in real-time in a range of live organisms and are providing novel insight into this dynamic process. Here, we highlight these methodological advances in intravital imaging of axonal transport, outlining key strengths and limitations while discussing findings, possible improvements, and outstanding questions.

  15. Phosphatidylserine improves axonal transport by inhibition of HDAC and has potential in treatment of neurodegenerative diseases

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    Shiran Naftelberg


    Full Text Available Familial dysautonomia (FD is a rare children neurodegenerative disease caused due to a point mutation in the IKBKAP gene that results in decreased IKK complex-associated protein (IKAP protein production. The disease affects mostly the dorsal root ganglion (DRG and the sympathetic ganglion. Recently, we found that the molecular mechanisms underlying neurodegeneration in FD patients are defects in axonal transport of nerve growth factors and microtubule stability in the DRG. Neurons are highly polarized cells with very long axons. In order to survive and maintain proper function, neurons depend on transport of proteins and other cellular components from the neuronal body along the axons. We further demonstrated that IKAP is necessary for axon maintenance and showed that phosphatidylserine acts as an HDAC6 inhibitor to rescue neuronal function in FD cells. In this review, we will highlight our latest research findings.

  16. Vesicular Axonal Transport is Modified In Vivo by Tau Deletion or Overexpression in Drosophila

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    Yasmina Talmat-Amar


    Full Text Available Structural microtubule associated protein Tau is found in high amount in axons and is involved in several neurodegenerative diseases. Although many studies have highlighted the toxicity of an excess of Tau in neurons, the in vivo understanding of the endogenous role of Tau in axon morphology and physiology is poor. Indeed, knock-out mice display no strong cytoskeleton or axonal transport phenotype, probably because of some important functional redundancy with other microtubule-associated proteins (MAPs. Here, we took advantage of the model organism Drosophila, which genome contains only one homologue of the Tau/MAP2/MAP4 family to decipher (endogenous Tau functions. We found that Tau depletion leads to a decrease in microtubule number and microtubule density within axons, while Tau excess leads to the opposite phenotypes. Analysis of vesicular transport in tau mutants showed altered mobility of vesicles, but no change in the total amount of putatively mobile vesicles, whereas both aspects were affected when Tau was overexpressed. In conclusion, we show that loss of Tau in tau mutants not only leads to a decrease in axonal microtubule density, but also impairs axonal vesicular transport, albeit to a lesser extent compared to the effects of an excess of Tau.

  17. Cryo Electron Tomography of Herpes Simplex Virus during Axonal Transport and Secondary Envelopment in Primary Neurons (United States)

    Ibiricu, Iosune; Huiskonen, Juha T.; Döhner, Katinka; Bradke, Frank; Sodeik, Beate; Grünewald, Kay


    During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the ‘married’ model or as non-enveloped capsids suggested by the ‘separate’ model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the ‘separate model’ for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles. PMID:22194682

  18. Effects of p-xylene inhalation on axonal transport in the rat retinal ganglion cells

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    Padilla, S.S.; Lyerly, D.P. (Environmental Protection Agency, Research Triangle Park, NC (USA))


    Although the solvent xylene is suspected of producing nervous system dysfunction in animals and humans, little is known regarding the neurochemical consequences of xylene inhalation. The intent of this study was to determine the effect of intermittent, acute, and subchronic p-xylene exposure on the axonal transport of proteins and glycoproteins within the rat retinofugal tract. A number of different exposure regimens were tested ranging from 50 ppm for a single 6-hr exposure to 1600 ppm 6 hr/day, 5 days/week, for a total of 8 exposure days. Immediately following removal from the inhalation chambers rats were injected intraocularly with (35S)methionine and (3H)fucose (to label retinal proteins and glycoproteins, respectively) and the axonal transport of labeled macromolecules to axons (optic nerve and optic tract) and nerve endings (lateral geniculate body and superior colliculus) was examined 20 hr after precursor injection. Only relatively severe exposure regimens (i.e., 800 or 1600 ppm 6 hr/day, 5 days/week, for 1.5 weeks) produced significant reductions in axonal transport; there was a moderate reduction in the axonal transport of 35S-labeled proteins in the 800-ppm-treated group which was more widespread in the 1600 ppm-treated group. Transport of 3H-labeled glycoproteins was less affected. Assessment of retinal metabolism immediately after isotope injection indicated that the rate of precursor uptake was not reduced in either treatment group. Furthermore, rapid transport was still substantially reduced in animals exposed to 1600 ppm p-xylene and allowed a 13-day withdrawal period. These data indicate that p-xylene inhalation decreases rapid axonal transport supplied to the projections of the rat retinal ganglion cells immediately after cessation of inhalation exposure and that this decreased transport is still apparent 13 days after the last exposure.

  19. Effects of p-xylene inhalation on axonal transport in the rat retinal ganglion cells

    International Nuclear Information System (INIS)

    Padilla, S.S.; Lyerly, D.P.


    Although the solvent xylene is suspected of producing nervous system dysfunction in animals and humans, little is known regarding the neurochemical consequences of xylene inhalation. The intent of this study was to determine the effect of intermittent, acute, and subchronic p-xylene exposure on the axonal transport of proteins and glycoproteins within the rat retinofugal tract. A number of different exposure regimens were tested ranging from 50 ppm for a single 6-hr exposure to 1600 ppm 6 hr/day, 5 days/week, for a total of 8 exposure days. Immediately following removal from the inhalation chambers rats were injected intraocularly with [35S]methionine and [3H]fucose (to label retinal proteins and glycoproteins, respectively) and the axonal transport of labeled macromolecules to axons (optic nerve and optic tract) and nerve endings (lateral geniculate body and superior colliculus) was examined 20 hr after precursor injection. Only relatively severe exposure regimens (i.e., 800 or 1600 ppm 6 hr/day, 5 days/week, for 1.5 weeks) produced significant reductions in axonal transport; there was a moderate reduction in the axonal transport of 35S-labeled proteins in the 800-ppm-treated group which was more widespread in the 1600 ppm-treated group. Transport of 3H-labeled glycoproteins was less affected. Assessment of retinal metabolism immediately after isotope injection indicated that the rate of precursor uptake was not reduced in either treatment group. Furthermore, rapid transport was still substantially reduced in animals exposed to 1600 ppm p-xylene and allowed a 13-day withdrawal period. These data indicate that p-xylene inhalation decreases rapid axonal transport supplied to the projections of the rat retinal ganglion cells immediately after cessation of inhalation exposure and that this decreased transport is still apparent 13 days after the last exposure

  20. Tri-partite complex for axonal transport drug delivery achieves pharmacological effect

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    Frederickson Martyn


    Full Text Available Abstract Background Targeted delivery of pharmaceutical agents into selected populations of CNS (Central Nervous System neurons is an extremely compelling goal. Currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. Systemic side effects or undesirable effects on parts of the CNS that are not involved in the pathology limit efficacy and limit clinical utility for many classes of pharmaceuticals. Axonal transport from the periphery offers a possible selective route, but there has been little progress towards design of agents that can accomplish targeted delivery via this intraneural route. To achieve this goal, we developed a tripartite molecular construction concept involving an axonal transport facilitator molecule, a polymer linker, and a large number of drug molecules conjugated to the linker, then sought to evaluate its neurobiology and pharmacological behavior. Results We developed chemical synthesis methodologies for assembling these tripartite complexes using a variety of axonal transport facilitators including nerve growth factor, wheat germ agglutinin, and synthetic facilitators derived from phage display work. Loading of up to 100 drug molecules per complex was achieved. Conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. Intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of CNS neurons. Pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a 300 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle. Conclusion Specific targeting of selected subpopulations of CNS neurons for drug delivery by axonal

  1. Cytoplasmic Dynein Transports Axonal Microtubules in a Polarity-Sorting Manner

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    Anand N. Rao


    Full Text Available Axonal microtubules are predominantly organized into a plus-end-out pattern. Here, we tested both experimentally and with computational modeling whether a motor-based polarity-sorting mechanism can explain this microtubule pattern. The posited mechanism centers on cytoplasmic dynein transporting plus-end-out and minus-end-out microtubules into and out of the axon, respectively. When cytoplasmic dynein was acutely inhibited, the bi-directional transport of microtubules in the axon was disrupted in both directions, after which minus-end-out microtubules accumulated in the axon over time. Computational modeling revealed that dynein-mediated transport of microtubules can establish and preserve a predominantly plus-end-out microtubule pattern as per the details of the experimental findings, but only if a kinesin motor and a static cross-linker protein are also at play. Consistent with the predictions of the model, partial depletion of TRIM46, a protein that cross-links axonal microtubules in a manner that influences their polarity orientation, leads to an increase in microtubule transport.

  2. Effect of the degree of polar mismatching on traffic jam formation in fast axonal transport. (United States)

    Kuznetsov, A V


    This paper simulates an axon with a region of reversed microtubule (MT) polarity, and investigates how the degree of polar mismatching in this region affects the formation of organelle traps in the axon. The model is based on modified Smith-Simmons equations governing molecular-motor-assisted transport in neurons. It is established that the structure that develops as a result of a region with disoriented MTs consists of two organelle traps, the trap to the left of this region accumulates plus-end-oriented organelles and the trap to the right of this region accumulates minus-end-oriented organelles. The presence of such a structure is shown to inhibit the transport of organelles down the axon. The degree by which the transport of organelles is inhibited depends on the degree of polar mismatching of MTs in the region between MT traps. Four cases with a different degree of polar mismatching are investigated.

  3. Sorting of cargos between axons and dendrites: modelling of differences in cargo transport in these two types of neurites. (United States)

    Kuznetsov, A V


    Explaining how intracellular cargos are sorted between axons and dendrites is important for a mechanistic understanding of what happens in many neurodegenerative disorders. A simple model of cargo sorting relies on differences in microtubule (MT) orientation between axons and dendrites: in mammalian neurons all MTs in axons have their plus ends directed outward while in proximal regions of dendrites the MT polarity is mixed. It can therefore be assumed that cargos that need to be driven into axons associate with kinesin motors while cargos that need to be driven into dendrites associate with dynein motors. This paper develops equations of cargo transport in axons and dendrites based on the above assumptions. Propagation of a pulse of radiolabelled cargos entering an axon and dendrite is simulated. The model equations are solved utilising the Laplace transform method. Differences in cargo transport between axons and dendrites are discussed.

  4. mRNP assembly, axonal transport, and local translation in neurodegenerative diseases. (United States)

    Khalil, Bilal; Morderer, Dmytro; Price, Phillip L; Liu, Feilin; Rossoll, Wilfried


    The development, maturation, and maintenance of the mammalian nervous system rely on complex spatiotemporal patterns of gene expression. In neurons, this is achieved by the expression of differentially localized isoforms and specific sets of mRNA-binding proteins (mRBPs) that regulate RNA processing, mRNA trafficking, and local protein synthesis at remote sites within dendrites and axons. There is growing evidence that axons contain a specialized transcriptome and are endowed with the machinery that allows them to rapidly alter their local proteome via local translation and protein degradation. This enables axons to quickly respond to changes in their environment during development, and to facilitate axon regeneration and maintenance in adult organisms. Aside from providing autonomy to neuronal processes, local translation allows axons to send retrograde injury signals to the cell soma. In this review, we discuss evidence that disturbances in mRNP transport, granule assembly, axonal localization, and local translation contribute to pathology in various neurodegenerative diseases, including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD). Copyright © 2018. Published by Elsevier B.V.

  5. Fast axonal transport of labeled proteins in motoneurons of exercise-trained rats

    International Nuclear Information System (INIS)

    Jasmin, B.J.; Lavoie, P.A.; Gardiner, P.F.


    In this study, the fast orthograde axonal transport of radiolabeled proteins was measured to determine the effects of endurance-running training on transport velocity and amounts of transported proteins in rat sciatic motoneurons. Female rats were subjected to a progressive running-training program for 10-12 wk. Twenty-four hours after the last training session, rats underwent right L4-L5 dorsal root ganglionectomy. The next day, 20 microCi of [3H]leucine was injected bilaterally in the vicinity of the motoneuronal cell bodies supplying the sciatic nerve, to study axonal transport parameters. Results showed that peak and average transport velocities of labeled proteins were significantly (P less than 0.05) increased by 22 and 29%, respectively, in the deafferented nerves of the runners as compared with controls. Moreover, the amount of total transported protein-bound radioactivity was increased in both left (40%) and right (37%) sciatic nerves of the runners. An exhaustive exercise session reduced (P less than 0.05) peak displacement (8%) and total transported protein-bound radioactivity (36%) in the sciatic nerves of control rats, whereas no changes were noticed in trained animals. The data suggest that chronic endurance running induces significant adaptations in the fast axonal transport of labeled proteins

  6. Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function. (United States)

    Otero, Maria G; Alloatti, Matías; Cromberg, Lucas E; Almenar-Queralt, Angels; Encalada, Sandra E; Pozo Devoto, Victorio M; Bruno, Luciana; Goldstein, Lawrence S B; Falzone, Tomás L


    Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

  7. Regulation of mitochondria-dynactin interaction and mitochondrial retrograde transport in axons. (United States)

    Drerup, Catherine M; Herbert, Amy L; Monk, Kelly R; Nechiporuk, Alex V


    Mitochondrial transport in axons is critical for neural circuit health and function. While several proteins have been found that modulate bidirectional mitochondrial motility, factors that regulate unidirectional mitochondrial transport have been harder to identify. In a genetic screen, we found a zebrafish strain in which mitochondria fail to attach to the dynein retrograde motor. This strain carries a loss-of-function mutation in actr10 , a member of the dynein-associated complex dynactin. The abnormal axon morphology and mitochondrial retrograde transport defects observed in actr10 mutants are distinct from dynein and dynactin mutant axonal phenotypes. In addition, Actr10 lacking the dynactin binding domain maintains its ability to bind mitochondria, arguing for a role for Actr10 in dynactin-mitochondria interaction. Finally, genetic interaction studies implicated Drp1 as a partner in Actr10-dependent mitochondrial retrograde transport. Together, this work identifies Actr10 as a factor necessary for dynactin-mitochondria interaction, enhancing our understanding of how mitochondria properly localize in axons.

  8. UV Irradiation Accelerates Amyloid Precursor Protein (APP) Processing and Disrupts APP Axonal Transport (United States)

    Almenar-Queralt, Angels; Falzone, Tomas L.; Shen, Zhouxin; Lillo, Concepcion; Killian, Rhiannon L.; Arreola, Angela S.; Niederst, Emily D.; Ng, Kheng S.; Kim, Sonia N.; Briggs, Steven P.; Williams, David S.


    Overexpression and/or abnormal cleavage of amyloid precursor protein (APP) are linked to Alzheimer's disease (AD) development and progression. However, the molecular mechanisms regulating cellular levels of APP or its processing, and the physiological and pathological consequences of altered processing are not well understood. Here, using mouse and human cells, we found that neuronal damage induced by UV irradiation leads to specific APP, APLP1, and APLP2 decline by accelerating their secretase-dependent processing. Pharmacological inhibition of endosomal/lysosomal activity partially protects UV-induced APP processing implying contribution of the endosomal and/or lysosomal compartments in this process. We found that a biological consequence of UV-induced γ-secretase processing of APP is impairment of APP axonal transport. To probe the functional consequences of impaired APP axonal transport, we isolated and analyzed presumptive APP-containing axonal transport vesicles from mouse cortical synaptosomes using electron microscopy, biochemical, and mass spectrometry analyses. We identified a population of morphologically heterogeneous organelles that contains APP, the secretase machinery, molecular motors, and previously proposed and new residents of APP vesicles. These possible cargoes are enriched in proteins whose dysfunction could contribute to neuronal malfunction and diseases of the nervous system including AD. Together, these results suggest that damage-induced APP processing might impair APP axonal transport, which could result in failure of synaptic maintenance and neuronal dysfunction. PMID:24573290

  9. Axonal transport of enzymes and labeled proteins in experimental axonopathy induced by p-bromophenylacetylurea

    International Nuclear Information System (INIS)

    Jakobsen, J.; Brimijoin, S.


    Axonal transport was studied by several techniques in the sciatic nerves of adult male Sprague-Dawley rats with neuropathy induced by treatment with p-bromophenylacetylurea (BPAU) in dimethylsulfoxide solution. Control rats were treated with solvent alone. BPAU, 200 mg/kg, induced severe muscle weakness in the hindlimbs, beginning after a latent period of 1 week and progressing to near total paralysis by 2 weeks. Axonal transport of the endogenous transmitter enzymes, acetylcholinesterase, dopamine-β-hydroxylase and choline acetyltransferase, was normal at both 2 and 15 days after administration of BPAU, as judged by the accumulation of enzyme activity above and below a set of double ligatures on the sciatic nerve. The velocity of fast anterograde transport of [ 35 S]methionine labeled protein was also unaffected by BPAU. However, 4 abnormalities of transport were detected in BPAU treated rats. These abnormalities are discussed. (Auth.)

  10. Phosphatidylserine Ameliorates Neurodegenerative Symptoms and Enhances Axonal Transport in a Mouse Model of Familial Dysautonomia.

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    Shiran Naftelberg


    Full Text Available Familial Dysautonomia (FD is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF. These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6 levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration.

  11. Investigating the Slow Axonal Transport of Neurofilaments: A Precursor for Optimal Neuronal Signaling (United States)

    Johnson, Christopher M.

    Neurofilaments are the intermediate filaments of neurons and are the most abundant structure of the neuronal cytoskeleton. Once synthesized within the cell body they are then transported throughout the axon along microtubule tracks, driven by the molecular motors kinesin and dynein. This movement is characterized by long pauses with no movement interrupted by infrequent bouts of rapid movement, resulting in an aggregate dense cytoskeletal structure, which serves to regulate an axon's shape and size. Curiously, the modulated kinetics of these polymers produces a very regular, yet non-uniform, morphology in myelinated axons which are composed of discretely spaced myelin-ensheathed segments that are separated by short constricted regions called "nodes of Ranvier". This unique design optimizes the conduction velocity of myelinated axons at minimal fiber size. Hence, neurofilaments regulate the axon caliber to optimize neuron function. The goal of this dissertation is to investigate the motile mechanism of neurofilament transport as well as the resulting electrophysiological effects that follow. We start by examining highly time-resolved kymograph images generated from recorded neurofilament movement via epifluorescence microscopy. Using kymograph analysis, edge detection algorithms, and pixel smoothing tactics, neurofilament trajectories are extracted and used to obtain statistical distributions for the characteristics of how these filaments move within cells. The results suggest that the observed intermittent and bidirectional motions of these filaments might be explained by a model in which dynein and kinesin motors attach to a single neurofilament cargo and interact through mechanical forces only (i.e. a "tug-of-war" model). We test this hypothesis by developing two discrete-state stochastic models for the kinetic cycles of kinesin and dynein, which are then incorporated into a separate stochastic model that represents the posed tug-of-war scenario. We then

  12. Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport

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    Martin Steuble


    Endocytosis of amyloid-β precursor protein (APP is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.

  13. Disruption of mitochondrial DNA replication in Drosophila increases mitochondrial fast axonal transport in vivo.

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    Rehan M Baqri

    Full Text Available Mutations in mitochondrial DNA polymerase (pol gamma cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol gamma leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol gamma deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol gamma-alpha mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.

  14. Quantification of retrograde axonal transport in the rat optic nerve by fluorogold spectrometry.

    Directory of Open Access Journals (Sweden)

    Christian van Oterendorp

    Full Text Available PURPOSE: Disturbed axonal transport is an important pathogenic factor in many neurodegenerative diseases, such as glaucoma, an eye disease characterised by progressive atrophy of the optic nerve. Quantification of retrograde axonal transport in the optic nerve usually requires labour intensive histochemical techniques or expensive equipment for in vivo imaging. Here, we report on a robust alternative method using Fluorogold (FG as tracer, which is spectrometrically quantified in retinal tissue lysate. METHODS: To determine parameters reflecting the relative FG content of a sample FG was dissolved in retinal lysates at different concentrations and spectra were obtained. For validation in vivo FG was injected uni- or bilaterally into the superior colliculus (SC of Sprague Dawley rats. The retinal lysate was analysed after 3, 5 and 7 days to determine the time course of FG accumulation in the retina (n = 15. In subsequent experiments axona transport was impaired by optic nerve crush (n = 3, laser-induced ocular hypertension (n = 5 or colchicine treatment to the SC (n = 10. RESULTS: Spectrometry at 370 nm excitation revealed two emission peaks at 430 and 610 nm. We devised a formula to calculate the relative FG content (c(FG, from the emission spectrum. c(FG is proportional to the real FG concentration as it corrects for variations of retinal protein concentration in the lysate. After SC injection, c(FG monotonously increases with time (p = 0.002. Optic nerve axonal damage caused a significant decrease of c(FG (crush p = 0.029; hypertension p = 0.025; colchicine p = 0.006. Lysates are amenable to subsequent protein analysis. CONCLUSIONS: Spectrometrical FG detection in retinal lysates allows for quantitative assessment of retrograde axonal transport using standard laboratory equipment. It is faster than histochemical techniques and may also complement morphological in vivo analyses.

  15. Subacute ethanol consumption reverses p-xylene-induced decreases in axonal transport

    Energy Technology Data Exchange (ETDEWEB)

    Padilla, S.; Lyerly, D.L.; Pope, C.N.


    Organic solvants, as a class, have been implicated as neurotoxic agents in humans and laboratory animals. The study was designed to assess the interaction between subacute ingestion of moderate levels of ethanol and the p-xylene-induced decreases in protein and glycoprotein synthesis and axonal transport in the rat optic system. The results indicated that animals maintained on 10% ethanol as a drinking liquid show less p-xylene-induced neurotoxicity than animals receiving no ethanol supplement.

  16. Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites (United States)

    Babic, Milos; Russo, Gary J.; Wellington, Andrea J.; Sangston, Ryan M.; Gonzalez, Migdalia


    Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state. PMID:25855186

  17. Cortical compression rapidly trimmed transcallosal projections and altered axonal anterograde transport machinery. (United States)

    Chen, Li-Jin; Wang, Yueh-Jan; Tseng, Guo-Fang


    Trauma and tumor compressing the brain distort underlying cortical neurons. Compressed cortical neurons remodel their dendrites instantly. The effects on axons however remain unclear. Using a rat epidural bead implantation model, we studied the effects of unilateral somatosensory cortical compression on its transcallosal projection and the reversibility of the changes following decompression. Compression reduced the density, branching profuseness and boutons of the projection axons in the contralateral homotopic cortex 1week and 1month post-compression. Projection fiber density was higher 1-month than 1-week post-compression, suggesting adaptive temporal changes. Compression reduced contralateral cortical synaptophysin, vesicular glutamate transporter 1 (VGLUT1) and postsynaptic density protein-95 (PSD95) expressions in a week and the first two marker proteins further by 1month. βIII-tubulin and kinesin light chain (KLC) expressions in the corpus callosum (CC) where transcallosal axons traveled were also decreased. Kinesin heavy chain (KHC) level in CC was temporarily increased 1week after compression. Decompression increased transcallosal axon density and branching profuseness to higher than sham while bouton density returned to sham levels. This was accompanied by restoration of synaptophysin, VGLUT1 and PSD95 expressions in the contralateral cortex of the 1-week, but not the 1-month, compression rats. Decompression restored βIII-tubulin, but not KLC and KHC expressions in CC. However, KLC and KHC expressions in the cell bodies of the layer II/III pyramidal neurons partially recovered. Our results show cerebral compression compromised cortical axonal outputs and reduced transcallosal projection. Some of these changes did not recover in long-term decompression. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Fluorescence Imaging of Fast Retrograde Axonal Transport in Living Animals

    Directory of Open Access Journals (Sweden)

    Dawid Schellingerhout


    Full Text Available Our purpose was to enable an in vivo imaging technology that can assess the anatomy and function of peripheral nerve tissue (neurography. To do this, we designed and tested a fluorescently labeled molecular probe based on the nontoxic C fragment of tetanus toxin (TTc. TTc was purified, labeled, and subjected to immunoassays and cell uptake assays. The compound was then injected into C57BL/6 mice (N = 60 for in vivo imaging and histologic studies. Image analysis and immunohistochemistry were performed. We found that TTc could be labeled with fluorescent moieties without loss of immunoreactivity or biologic potency in cell uptake assays. In vivo fluorescent imaging experiments demonstrated uptake and retrograde transport of the compound along the course of the sciatic nerve and in the spinal cord. Ex vivo imaging and immunohistochemical studies confirmed the presence of TTc in the sciatic nerve and spinal cord, whereas control animals injected with human serum albumin did not exhibit these features. We have demonstrated neurography with a fluorescently labeled molecular imaging contrast agent based on the TTc.

  19. Extracellular Tau Oligomers Induce Invasion of Endogenous Tau into the Somatodendritic Compartment and Axonal Transport Dysfunction (United States)

    Swanson, Eric; Breckenridge, Leigham; McMahon, Lloyd; Som, Sreemoyee; McConnell, Ian; Bloom, George S.


    Aggregates composed of the microtubule associated protein, tau, are a hallmark of Alzheimer’s disease and non-Alzheimer’s tauopathies. Extracellular tau can induce the accumulation and aggregation of intracellular tau, and tau pathology can be transmitted along neural networks over time. There are six splice variants of central nervous system tau, and various oligomeric and fibrillar forms are associated with neurodegeneration in vivo. The particular extracellular forms of tau capable of transferring tau pathology from neuron to neuron remain ill defined, however, as do the consequences of intracellular tau aggregation on neuronal physiology. The present study was undertaken to compare the effects of extracellular tau monomers, oligomers, and filaments comprising various tau isoforms on the behavior of cultured neurons. We found that 2N4R or 2N3R tau oligomers provoked aggregation of endogenous intracellular tau much more effectively than monomers or fibrils, or of oligomers made from other tau isoforms, and that a mixture of all six isoforms most potently provoked intracellular tau accumulation. These effects were associated with invasion of tau into the somatodendritic compartment. Finally, we observed that 2N4R oligomers perturbed fast axonal transport of membranous organelles along microtubules. Intracellular tau accumulation was often accompanied by increases in the run length, run time and instantaneous velocity of membranous cargo. This work indicates that extracellular tau oligomers can disrupt normal neuronal homeostasis by triggering axonal tau accumulation and loss of the polarized distribution of tau, and by impairing fast axonal transport. PMID:28482642

  20. NAP (davunetide) modifies disease progression in a mouse model of severe neurodegeneration: protection against impairments in axonal transport. (United States)

    Jouroukhin, Yan; Ostritsky, Regina; Assaf, Yaniv; Pelled, Galit; Giladi, Eliezer; Gozes, Illana


    NAP (davunetide) is a novel neuroprotective compound with mechanism of action that appears to involve microtubule (MT) stabilization and repair. To evaluate, for the first time, the impact of NAP on axonal transport in vivo and to translate it to neuroprotection in a severe neurodegeneration, the SOD1-G93A mouse model for amyotrophic lateral sclerosis (ALS) was used. Manganese-enhanced magnetic resonance imaging (MRI), estimating axonal transport rates, revealed a significant reduction of the anterograde axonal transport in the ALS mice compared to healthy control mice. Acute NAP treatment normalized axonal transport rates in these ALS mice. Tau hyperphosphorylation, associated with MT dysfunction and defective axonal transport, was discovered in the brains of the ALS mice and was significantly reduced by chronic NAP treatment. Furthermore, in healthy wild type (WT) mice, NAP reversed axonal transport disruption by colchicine, suggesting drug-dependent protection against axonal transport impairment through stabilization of the neuronal MT network. Histochemical analysis showed that chronic NAP treatment significantly protected spinal cord motor neurons against ALS-like pathology. Sequential MRI measurements, correlating brain structure with ALS disease progression, revealed a significant damage to the ventral tegmental area (VTA), indicative of impairments to the dopaminergic pathways relative to healthy controls. Chronic daily NAP treatment of the SOD1-G93A mice, initiated close to disease onset, delayed degeneration of the trigeminal, facial and hypoglossal motor nuclei as was significantly apparent at days 90-100 and further protected the VTA throughout life. Importantly, protection of the VTA was significantly correlated with longevity and overall, NAP treatment significantly prolonged life span in the ALS mice. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Fast axonal transport of 3H-leucin-labelled proteins in the unhurt and isolated optical nerve of rats

    International Nuclear Information System (INIS)

    Wagner, H.E.


    The distribution of radioactivity of amino acid molecules incorporated in protein after injection of 3 H-Leucin into the right bulb was investigated and determined along optical nerve after 1, 2, and 4 h. A slightly increased radioactivity at the point of entrance of the optical nerves into the optical duct was found. A slightly reduced axon diameter was discussed as a possible cause. The radioactivity brought into the optical nerve via the vascular system was determined by measuring the contralateral optical nerve. In relation to the axonally transported activity, it was low. The speed of the fast axonal transport is 168 mm/d. If the processes ruling the amino acids in the perikaryon are taken into consideration, the transport speed is 240 mm/d. The application of the protein synthesis prohibitor, Cycloheximide, 5 minutes after the injection of Leucinin completely prevented the appearance of axonally transported labelled proteins. When cycloheximide was administered 2 h after Leucin, a significantly loner radioactivity than in the nerve could be determined after another 2 h; i.e. the incorporation of Leucin was not completed yet after 2 h. The profile of active compounds was the same as in the control group. In other experiments, the axonal transport of labelled proteins in isolated optical nerve fibres was tested. If the separation was carried out 2 h after the injection of Leucin an extreme reduction in activity could be determined after 1 or 2 h. The continued distribution of activity after cycloheximide treatment and removal of perikarya in comparison with the control indicate the continuation of the transport, also after separation of the axon from the perikaryon. This means that, during the time of the experiment, the mechanism of the fast axonal transport functions independently of the perikaryon. (orig./MG) [de

  2. A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Wendy M. Knowlton


    Full Text Available The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo, we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1. Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7, and isp-1, and the putative oxidoreductase rad-8. In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1. Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or

  3. Herpes simplex virus gE/gI extracellular domains promote axonal transport and spread from neurons to epithelial cells. (United States)

    Howard, Paul W; Wright, Catherine C; Howard, Tiffani; Johnson, David C


    Following reactivation from latency, there are two distinct steps in the spread of herpes simplex virus (HSV) from infected neurons to epithelial cells: (i) anterograde axonal transport of virus particles from neuron bodies to axon tips and (ii) exocytosis and spread of extracellular virions across cell junctions into adjacent epithelial cells. The HSV heterodimeric glycoprotein gE/gI is important for anterograde axonal transport, and gE/gI cytoplasmic domains play important roles in sorting of virus particles into axons. However, the roles of the large (∼400-residue) gE/gI extracellular (ET) domains in both axonal transport and neuron-to-epithelial cell spread have not been characterized. Two gE mutants, gE-277 and gE-348, contain small insertions in the gE ET domain, fold normally, form gE/gI heterodimers, and are incorporated into virions. Both gE-277 and gE-348 did not function in anterograde axonal transport; there were markedly reduced numbers of viral capsids and glycoproteins compared with wild-type HSV. The defects in axonal transport were manifest in neuronal cell bodies, involving missorting of HSV capsids before entry into proximal axons. Although there were diminished numbers of mutant gE-348 capsids and glycoproteins in distal axons, there was efficient spread to adjacent epithelial cells, similar to wild-type HSV. In contrast, virus particles produced by HSV gE-277 spread poorly to epithelial cells, despite numbers of virus particles similar to those for HSV gE-348. These results genetically separate the two steps in HSV spread from neurons to epithelial cells and demonstrate that the gE/gI ET domains function in both processes. An essential phase of the life cycle of herpes simplex virus (HSV) and other alphaherpesviruses is the capacity to reactivate from latency and then spread from infected neurons to epithelial tissues. This spread involves at least two steps: (i) anterograde transport to axon tips followed by (ii) exocytosis and extracellular

  4. Ndel1-derived peptides modulate bidirectional transport of injected beads in the squid giant axon

    Directory of Open Access Journals (Sweden)

    Michal Segal


    Bidirectional transport is a key issue in cellular biology. It requires coordination between microtubule-associated molecular motors that work in opposing directions. The major retrograde and anterograde motors involved in bidirectional transport are cytoplasmic dynein and conventional kinesin, respectively. It is clear that failures in molecular motor activity bear severe consequences, especially in the nervous system. Neuronal migration may be impaired during brain development, and impaired molecular motor activity in the adult is one of the hallmarks of neurodegenerative diseases leading to neuronal cell death. The mechanisms that regulate or coordinate kinesin and dynein activity to generate bidirectional transport of the same cargo are of utmost importance. We examined how Ndel1, a cytoplasmic dynein binding protein, may regulate non-vesicular bidirectional transport. Soluble Ndel1 protein, Ndel1-derived peptides or control proteins were mixed with fluorescent beads, injected into the squid giant axon, and the bead movements were recorded using time-lapse microscopy. Automated tracking allowed for extraction and unbiased analysis of a large data set. Beads moved in both directions with a clear bias to the anterograde direction. Velocities were distributed over a broad range and were typically slower than those associated with fast vesicle transport. Ironically, the main effect of Ndel1 and its derived peptides was an enhancement of anterograde motion. We propose that they may function primarily by inhibition of dynein-dependent resistance, which suggests that both dynein and kinesin motors may remain engaged with microtubules during bidirectional transport.

  5. Effects of kainic acid lesions in lateral geniculate nucleus: activity dependence of retrograde axonal transport of fluorescent dyes. (United States)

    Woodward, W R; Coull, B M


    Kainic acid lesions in the dorsal lateral geniculate nucleus of rats block the retrograde axonal transport of fluorescent dyes in corticogeniculate neurons without affecting the retrograde transport of D-aspartate or the orthograde transport of radiolabelled proteins in these neurons. This blocking of dye transport does not appear to be a consequence of kainic acid-induced damage to axon terminals in the geniculate since retinal ganglion cells are still able to transport dyes retrograde. A more likely explanation for these results is that fluorescent dye transport requires electrical activity in neurons, and elimination of the geniculate afferents to visual cortex reduces impulse traffic in cortical output fibers to a level below that required to support detectable dye transport. This interpretation is supported by the observation that kainic acid lesions also reduce retrograde transport of dyes in cortical neurons which project to the superior colliculus. Electrical stimulation in the subcortical white matter restores the transport of dye compounds in corticogeniculate neurons: evidence consistent with an activity-dependent mechanism of retrograde transport for these substances. These results provide evidence that axon terminals of retinal ganglion cells and corticogeniculate neurons survive in kainate-lesioned geniculates and are capable of normal neuronal function.

  6. Regulation of Auxin Transport by Phosphorylation and Flavonoids during Gravitropism in Arabidopsis (United States)

    Muday, Gloria K.


    The focus of this research includes: 1) Regulation of Axin transport by flavonoids during gravitropism; 2) Phosphorylation control of auxin transport during gravity response; 3) Ethylene regulation of gravitropic curvature; 4) IBA transport and gravitropic response; and 5) Other collaborative projects.

  7. Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain.

    Directory of Open Access Journals (Sweden)

    Hiroaki Mochizuki


    Full Text Available Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II, an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM and No distributive disjunction (Nod, remains unaltered. Genetic analyses of kinesin light chain (Klc and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of

  8. Axonal collateral-collateral transport of tract tracers in brain neurons: false anterograde labelling and useful tool. (United States)

    Chen, S; Aston-Jones, G


    It is well established that some neuroanatomical tracers may be taken up by local axonal terminals and transported to distant axonal collaterals (e.g., transganglionic transport in dorsal root ganglion cells). However, such collateral-collateral transport of tracers has not been systematically examined in the central nervous system. We addressed this issue with four neuronal tracers--biocytin, biotinylated dextran amine, cholera toxin B subunit, and Phaseolus vulgaris-leucoagglutinin--in the cerebellar cortex. Labelling of distant axonal collaterals in the cerebellar cortex (indication of collateral-collateral transport) was seen after focal iontophoretic microinjections of each of the four tracers. However, collateral-collateral transport properties differed among these tracers. Injection of biocytin or Phaseolus vulgaris-leucoagglutinin in the cerebellar cortex yielded distant collateral labelling only in parallel fibres. In contrast, injection of biotinylated dextran amine or cholera toxin B subunit produced distant collateral labelling of climbing fibres and mossy fibres, as well as parallel fibres. The present study is the first systematic examination of collateral-collateral transport following injection of anterograde tracers in brain. Such collateral-collateral transport may produce false-positive conclusions regarding neural connections when using these tracers for anterograde transport. However, this property may also be used as a tool to determine areas that are innervated by common distant afferents. In addition, these results may indicate a novel mode of chemical communication in the nervous system.

  9. Influence of ionizing radiation on the rate of substance axon transport and its hormone-stimulated increase in rat nerves

    International Nuclear Information System (INIS)

    Frol'kyis, V.V.; Tanin, S.A.; Martsinko, V.Yi.; Gorban', Je.M.


    The influence of x-ray on the rate of substance axon transport (AT) and its hormone-stimulated increase was studied. The study involved 80 Wistar male rats aged 8 - 10 months. Post-irradiation reduction of AT rate is one of the factors, forming trophic disturbances, while anabolic hormones can prevent them

  10. Modeling Huntington disease in Drosophila: Insights into axonal transport defects and modifiers of toxicity. (United States)

    Krench, Megan; Littleton, J Troy


    Huntington disease (HD) is an inherited neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the huntingtin (Htt) gene. Despite years of research, there is no treatment that extends life for patients with the disorder. Similarly, little is known about which cellular pathways that are altered by pathogenic Huntingtin (Htt) protein expression are correlated with neuronal loss. As part of a longstanding effort to gain insights into HD pathology, we have been studying the protein in the context of the fruitfly Drosophila melanogaster. We generated transgenic HD models in Drosophila by engineering flies that carry a 12-exon fragment of the human Htt gene with or without the toxic trinucleotide repeat expansion. We also created variants with a monomeric red fluorescent protein (mRFP) tag fused to Htt that allows in vivo imaging of Htt protein localization and aggregation. While wild-type Htt remains diffuse throughout the cytoplasm of cells, pathogenic Htt forms insoluble aggregates that accumulate in neuronal soma and axons. Aggregates can physically block transport of numerous organelles along the axon. We have also observed that aggregates are formed quickly, within just a few hours of mutant Htt expression. To explore mechanisms of neurodegeneration in our HD model, we performed in vivo and in vitro screens to search for modifiers of viability and pathogenic Htt aggregation. Our results identified several novel candidates for HD therapeutics that can now be tested in mammalian models of HD. Furthermore, these experiments have highlighted the complex relationship between aggregates and toxicity that exists in HD.

  11. Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases (United States)

    Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali


    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

  12. Disruption of axonal transport perturbs bone morphogenetic protein (BMP)--signaling and contributes to synaptic abnormalities in two neurodegenerative diseases. (United States)

    Kang, Min Jung; Hansen, Timothy J; Mickiewicz, Monique; Kaczynski, Tadeusz J; Fye, Samantha; Gunawardena, Shermali


    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.

  13. Characterization of intracellular regions in the human serotonin transporter for phosphorylation sites

    DEFF Research Database (Denmark)

    Sørensen, Lena; Strømgaard, Kristian; Kristensen, Anders S


    /dephosphorylation during transporter regulation by multiple pathways. In particular, activation and/or inhibition of kinases including PKC, PKG, p38MAPK, and CaMKII modulate SERT function and trafficking. The molecular mechanisms by which kinase activity is linked to SERT regulation are poorly understood, including...... the identity of specific phosphorylated residues. To elucidate SERT phosphorylation sites, we have generated peptides corresponding to the entire intracellular region of human SERT and performed in vitro phosphorylation assays with a panel of kinases suggested to be involved in SERT regulation or for which...

  14. Increased slow transport in axons of regenerating newt limbs after a nerve conditioning lesion made prior to amputation

    Energy Technology Data Exchange (ETDEWEB)

    Maier, C.E.


    The first part of this study shows that axonal density is constant in the limb stump of the next proximal to the area of traumatic nerve degeneration caused by limb amputation. The results of the second part of this work reveal that a nerve conditioning lesion made two weeks prior to amputation is associated with accelerated limb regeneration and that this accelerated limb regeneration is accompanied by an earlier arrival of axons. This is the first demonstration of naturally occurring limb regeneration being enhanced. In this study SCb cytoskeletal proteins were identified and measured using SDS-PAGE and liquid scintillation counting. Proteins were measured at 7, 14, 21, and 28 days after {sup 35}S-methionine injection and the normal rate of SCb transport determined to be 0.19 mm/day. A single axotomy does not enhance the rate of SCb transport but does increase the amount of labeled SCb proteins that are transported. When a conditioning lesion is employed prior to limb amputation and SCb proteins are measured at 7, 14, and 21 days after injection, there is a twofold acceleration in the rate of SCb transport and an increase in the amount of SCb proteins transported in conditioned axons.

  15. Dissociation of insulin receptor phosphorylation and stimulation of glucose transport in BC3H-1 myocytes

    International Nuclear Information System (INIS)

    Mojsilovic, L.P.; Standaert, M.L.; Rosic, N.K.; Pollet, R.J.


    The authors have investigated insulin receptor phosphorylation in differentiated cultured BC3H-1 myocytes. As for other insulin-responsive cell systems in partially purified wheat germ agglutinin receptor preparations, insulin stimulates the phosphorylation of its own receptor (95K β-subunits) in a dose dependent manner (0-400 nM), as identified by immunoprecipitation with antiinsulin receptor antibodies and SDS-PAGE. In the same preparations they show that 12-0-tetradecanyl phorbol acetate (TPA), which in many respect β-subunits in the same dose dependent manner (0-5 μM). In addition, antiinsulin receptor antibodies (B-10) also induced phosphorylation of mimics insulin action, also induced phosphorylation of the insulin receptor and HPLC tryptic maps of the 32 P-labeled β-subunit were identical to those for insulin-induced receptor phosphorylation. However, while insulin and TPA are potent stimulators of glucose transport in these muscle cells, the antireceptor antibodies alone failed to provoke glucose transport at any concentration. The specificity and activity of these antibodies were confirmed in their system by their ability to inhibit insulin binding and insulin-stimulated glucose transport in a concentration-dependent manner. Their results indicate that phosphorylation of insulin receptor is not a crucial event in mediating insulin action, at least with respect to glucose transport. While the effects of the B-10 antibody in the BC3H-1 myocyte differ from those in the adipocyte, their results provide independent confirmation of their essential conclusion that phosphorylation of the insulin receptor may not be necessary nor sufficient for its acute action in promoting glucose transport

  16. Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides : The coupling between transport and phosphorylation in inside-out vesicles

    NARCIS (Netherlands)

    Lolkema, Juke S.; Robillard, George T.

    The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the

  17. Limited trafficking of a neurotropic virus through inefficient retrograde axonal transport and the type I interferon response.

    Directory of Open Access Journals (Sweden)

    Karen Z Lancaster


    Full Text Available Poliovirus is an enteric virus that rarely invades the human central nervous system (CNS. To identify barriers limiting poliovirus spread from the periphery to CNS, we monitored trafficking of 10 marked viruses. After oral inoculation of susceptible mice, poliovirus was present in peripheral neurons, including vagus and sciatic nerves. To model viral trafficking in peripheral neurons, we intramuscularly injected mice with poliovirus, which follows a muscle-sciatic nerve-spinal cord-brain route. Only 20% of the poliovirus population successfully moved from muscle to brain, and three barriers limiting viral trafficking were identified. First, using light-sensitive viruses, we found limited viral replication in peripheral neurons. Second, retrograde axonal transport of poliovirus in peripheral neurons was inefficient; however, the efficiency was increased upon muscle damage, which also increased the transport efficiency of a non-viral neural tracer, wheat germ agglutinin. Third, using susceptible interferon (IFN alpha/beta receptor knockout mice, we demonstrated that the IFN response limited viral movement from the periphery to the brain. Surprisingly, the retrograde axonal transport barrier was equivalent in strength to the IFN barrier. Illustrating the importance of barriers created by the IFN response and inefficient axonal transport, IFN alpha/beta receptor knockout mice with muscle damage permitted 80% of the viral population to access the brain, and succumbed to disease three times faster than mice with intact barriers. These results suggest that multiple separate barriers limit poliovirus trafficking from peripheral neurons to the CNS, possibly explaining the rare incidence of paralytic poliomyelitis. This study identifies inefficient axonal transport as a substantial barrier to poliovirus trafficking in peripheral neurons, which may limit CNS access for other viruses.

  18. [The effect of hormones on the rate of axonal transport in the ventral spinal nerve roots of rats]. (United States)

    Frol'kis, V V; Tanin, S A; Martsinko, V I


    The Wistar male rats in the age of 8-12 months were injected 7-8 microliter of aqueous solution of L-leucine-14C (specific activity 12543 megaBq/mmol) into the area of the ventral horn at the level of L5,6 segment of the spinal cord. The study of radioactivity in various sections of the respective frontal root was performed after one hour. It was found that estradiol dipropionate, testosterone propionate, insulin and small doses of thyroxin increased the axonal transport of the labelled material, while hydrocortisone, large doses of thyroxin, castration and thyroidectomy caused its delay. It is concluded that the axonal transport is under a pronounced hormonal control.

  19. Microtubule-stabilizing peptides and small molecules protecting axonal transport and brain function: focus on davunetide (NAP). (United States)

    Magen, Iddo; Gozes, Illana


    This review focuses on the therapeutic effects and mechanisms of action of NAP (davunetide), an eight amino acid snippet derived from activity-dependent neuroprotective protein (ADNP) which was discovered in our laboratory. We have recently described the effects of NAP in neurodegenerative disorders, and we now review the beneficial effects of NAP and other microtubule-stabilizing agents on impairments in axonal transport. Experiments in animal models of microtubule-deficiency including tauopathy (spanning from drosophila to mammals) showed protection of axonal transport by microtubule-stabilizers and NAP, which was coupled to motor and cognitive protection. Clinical trials with NAP (davunetide) are reviewed paving the path to future developments. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Axonal transport in rats rendered paraplegic following a single subarachnoid injection of either batrachotoxin or 6-aminonicotinamide into the spinal cord. (United States)

    Boegman, R J; Albuquerque, E X


    Batrachotoxin (BTX) or 6-aminonicotinamide (6-AN) when injected into the subarachnoidal space of the lumbar spinal cord block fast axonal transport of 3H-protein in motor nerves. Axonal transport recovers partially within one day after administering BTX while the effect of 6-AN lasts for more than 21 days. These observations are discussed in relation to the onset and recovery of membrane depolarization observed in the extensor muscle.

  1. Phosphorylation is required for the pathogen defense function of the Arabidopsis PEN3 ABC transporter (United States)

    The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against multiple pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whet...

  2. Aberrant GlyRS-HDAC6 interaction linked to axonal transport deficits in Charcot-Marie-Tooth neuropathy. (United States)

    Mo, Zhongying; Zhao, Xiaobei; Liu, Huaqing; Hu, Qinghua; Chen, Xu-Qiao; Pham, Jessica; Wei, Na; Liu, Ze; Zhou, Jiadong; Burgess, Robert W; Pfaff, Samuel L; Caskey, C Thomas; Wu, Chengbiao; Bai, Ge; Yang, Xiang-Lei


    Dominant mutations in glycyl-tRNA synthetase (GlyRS) cause a subtype of Charcot-Marie-Tooth neuropathy (CMT2D). Although previous studies have shown that GlyRS mutants aberrantly interact with Nrp1, giving insight into the disease's specific effects on motor neurons, these cannot explain length-dependent axonal degeneration. Here, we report that GlyRS mutants interact aberrantly with HDAC6 and stimulate its deacetylase activity on α-tubulin. A decrease in α-tubulin acetylation and deficits in axonal transport are observed in mice peripheral nerves prior to disease onset. An HDAC6 inhibitor used to restore α-tubulin acetylation rescues axonal transport deficits and improves motor functions of CMT2D mice. These results link the aberrant GlyRS-HDAC6 interaction to CMT2D pathology and suggest HDAC6 as an effective therapeutic target. Moreover, the HDAC6 interaction differs from Nrp1 interaction among GlyRS mutants and correlates with divergent clinical presentations, indicating the existence of multiple and different mechanisms in CMT2D.

  3. Secretagogue-induced protein phosphorylation and chloride transport in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Burnham, D.B.; Fondacaro, J.D.


    The effects of vasoactive intestinal polypeptide (VIP), 16,16-dimethyl prostaglandin E2 (DMPGE2) and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on protein phosphorylation were studied in relation to stimulation of chloride transport in cell suspensions of the human colon epithelial cell line Caco-2. In /sup 36/Cl-loaded cells, VIP and DMPGE2 within 1 min decreased cellular chloride content 35-40%, with half-maximal effects being elicited at 1.0 and 85 nM concentration, respectively. A similar effect on chloride content occurred after 10 min of treatment with 0.5 mM DBcAMP. For all three secretagogues, decreases in cellular chloride content were associated with increases in membrane permeability to chloride. DMPGE2 and VIP within 1 min, and DBcAMP within 10 min, increased the phosphorylation of an unidentified soluble protein of Mr = 42,000 and pI = 6.1, and of a protein of Mr = 20,200 and pI = 4.9 identified as myosin regulatory light chain. Between 10 and 30 min of stimulation, however, phosphorylation of the Mr = 42,000 protein and chloride transport activity remained elevated in DMPGE2- and DBcAMP-treated cells, whereas light chain phosphorylation returned to control level. No effect of secretagogues on phosphorylation was detected in the total particulate fraction or an integral membrane protein fraction. It is concluded that increased membrane permeability to chloride induced by cAMP-mediated secretagogues in Caco-2 is temporally associated with the increased phosphorylation of a Mr = 42,000 soluble protein.

  4. Peculiarities of axonal transport of steroid hormones (hydrocortisone, testosterone) in spinal root fibres of adult and old rats. (United States)

    Frolkis, V V; Tanin, S A


    The labelled steroid hormones [3H]hydrocortisone and [14C]testosterone, being injected into the gray matter of the rat spinal cord L5-L6 segments, were shown to be transported at a high velocity along the ventral (anterograde) and dorsal (retrograde) root fibres. The maximum velocity of axonal transport along the ventral and dorsal roots in adult rats was, on average, 3006 +/- 101 and 3028 +/- 48 mm/day for [3H]hydrocortisone and 4594 +/- 186 and 5185 +/- 485 mm/day for [14C]testosterone, respectively. In old rats, axonal transport of steroid hormones was markedly slower. Its maximum velocity along the ventral and dorsal roots averaged to 756 +/- 64 and 738 +/- 48 mm/day for [3H]hydrocortisone and 624 +/- 54 and 608 +/- 80 mm/day for [14C]testosterone, respectively. In old rats the amount of labelled hydrocortisone incorporated into the ventral root fibres was sharply reduced (by more than an order of the value) as compared to that in adult animals. At the same time, the intensity of the labelled testosterone incorporation into the ventral root fibres did not demonstrate any significant age-related difference. The injection of low doses of steroid hormones (from less than one microgram to a few micrograms) into the lumbar spinal cord resulted in a significant hyperpolarization several hours later first of the gastrocnemius and then of deltoideus muscle fibres. In old rats, such a hyperpolarization occurred much later. It is suggested that axonal transport of steroid hormones is one of the mechanisms responsible for the effects of hormones on the tissues, which undergoes considerable changes with ageing.

  5. Axoplasmic transport of substances in motoneuronal axons of the spinal cord in old age. (United States)

    Frolkis, V V; Tanin, S A; Marcinko, V I; Kulchitsky, O K; Yasechko, A V


    Seven to eight microliters of aqueous solution of L-[14C]Leucine (spec. act. 339 mCi/mmol) were introduced in the zone of ventral horn (L5-L6) of the spinal cord of adult (8-12 months) and old (26-28 months) rats. The radioactivity of various parts of the corresponding ventral roots was measured 1-2.5 h thereafter. Labelled substances (including protein) were found to migrate with fast flow in adult rats at the rate of 408 +/- 10.9 and 380 +/- 22 mm/24 h, respectively, as compared with 217 +/- 11.3 and 200 +/- 40 mm/24 h in old rats. The axoplasmic flow slows down in old rats with the increase of distance from the neuronal body. Uncoupling of oxidation and phosphorylation by the administration of 2,4-dinitrophenol, inhibition of glycolysis with NaF, hypoxemia produce more marked deceleration of axoplasmic flow in old rats, while small doses of NaF accelerate the flow, which correlates with the rise of cAMP in ventral roots. Sex steroids accelerate significantly the rate of axoplasmic flow. There is a marked increase in the rate due to the administration of estradiol dipropionate in old rats and due to testosterone propionate in adult animals. Changes in resting membrane potential and direct excitability thresholds of some muscles following colchicine blockade of axoplasmic transport are less marked in old rats that evidences for the weakening of neurotrophic control in old age.

  6. Analytical comparison between Nixon-Logvinenko's and Jung-Brown's theories of slow neurofilament transport in axons. (United States)

    Kuznetsov, I A; Kuznetsov, A V


    This paper develops analytical solutions describing slow neurofilament (NF) transport in axons. The obtained solutions are based on two theories of NF transport: Nixon-Logvinenko's theory that postulates that most NFs are incorporated into a stationary cross-linked network and only a small pool is slowly transported and Jung-Brown's theory that postulates a single dynamic pool of NFs that are transported according to the stop-and-go hypothesis. The simplest two-kinetic state version of the model developed by Jung and Brown was compared with the theory developed by Nixon and Logvinenko. The model for Nixon-Logvinenko's theory included stationary, pausing, and running NF populations while the model used for Jung-Brown's theory only included pausing and running NF populations. Distributions of NF concentrations resulting from Nixon-Logvinenko's and Jung-Brown's theories were compared. In previous publications, Brown and colleagues successfully incorporated slowing of NF transport into their model by assuming that some kinetic constants depend on the distance from the axon hillock. In this paper we defined the average rate of NF transport as the rate of motion of the center of mass of radiolabeled NFs. We have shown that for this definition, if all kinetic rates are assumed constant, Jung-Brown's theory predicts a constant average rate of NF transport. We also demonstrated that Nixon-Logvinenko's theory predicts slowing of NF transport even if all kinetic rates are assumed constant, and the obtained slowing agrees well with published experimental data. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Protein synthesis in axons and terminals: significance for maintenance, plasticity and regulation of phenotype. With a critique of slow transport theory. (United States)

    Alvarez, J; Giuditta, A; Koenig, E


    This article focuses on local protein synthesis as a basis for maintaining axoplasmic mass, and expression of plasticity in axons and terminals. Recent evidence of discrete ribosomal domains, subjacent to the axolemma, which are distributed at intermittent intervals along axons, are described. Studies of locally synthesized proteins, and proteins encoded by RNA transcripts in axons indicate that the latter comprise constituents of the so-called slow transport rate groups. A comprehensive review and analysis of published data on synaptosomes and identified presynaptic terminals warrants the conclusion that a cytoribosomal machinery is present, and that protein synthesis could play a role in long-term changes of modifiable synapses. The concept that all axonal proteins are supplied by slow transport after synthesis in the perikaryon is challenged because the underlying assumptions of the model are discordant with known metabolic principles. The flawed slow transport model is supplanted by a metabolic model that is supported by evidence of local synthesis and turnover of proteins in axons. A comparison of the relative strengths of the two models shows that, unlike the local synthesis model, the slow transport model fails as a credible theoretical construct to account for axons and terminals as we know them. Evidence for a dynamic anatomy of axons is presented. It is proposed that a distributed "sprouting program," which governs local plasticity of axons, is regulated by environmental cues, and ultimately depends on local synthesis. In this respect, nerve regeneration is treated as a special case of the sprouting program. The term merotrophism is proposed to denote a class of phenomena, in which regional phenotype changes are regulated locally without specific involvement of the neuronal nucleus.

  8. ATM and GLUT1-S490 phosphorylation regulate GLUT1 mediated transport in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Stanley Andrisse

    Full Text Available The glucose and dehydroascorbic acid (DHA transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM. The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1.L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity.In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1, which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL.These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction.

  9. Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis

    International Nuclear Information System (INIS)

    Martin, S.A.; Russell, J.B.


    Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments, indicated that separate phosphotransferases systems existed for glucose, maltose, and sucrose. [ 14 C]maltose transport was inhibited by excess glucose and to a lesser extent by sucrose. [ 14 C]glucose and [ 14 C]sucrose transports were not inhibited by an excess of maltose. Since [ 14 C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of P/sub i/ was increased from 0 to 100 mM, a maltose phosphorylase was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for α-glucose 1-phosphate. Only sucrose-grown cells possessed sucrose hydrolase activity, and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities

  10. Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules. (United States)

    Jorge-Finnigan, Ana; Kleppe, Rune; Jung-Kc, Kunwar; Ying, Ming; Marie, Michael; Rios-Mondragon, Ivan; Salvatore, Michael F; Saraste, Jaakko; Martinez, Aurora


    Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. A cAMP/PKA/Kinesin-1 Axis Promotes the Axonal Transport of Mitochondria in Aging Drosophila Neurons. (United States)

    Vagnoni, Alessio; Bullock, Simon L


    Mitochondria play fundamental roles within cells, including energy provision, calcium homeostasis, and the regulation of apoptosis. The transport of mitochondria by microtubule-based motors is critical for neuronal structure and function. This process allows local requirements for mitochondrial functions to be met and also facilitates recycling of these organelles [1, 2]. An age-related reduction in mitochondrial transport has been observed in neurons of mammalian and non-mammalian organisms [3-6], and has been proposed to contribute to the broader decline in neuronal function that occurs during aging [3, 5-7]. However, the factors that influence mitochondrial transport in aging neurons are poorly understood. Here we provide evidence using the tractable Drosophila wing nerve system that the cyclic AMP/protein kinase A (cAMP/PKA) pathway promotes the axonal transport of mitochondria in adult neurons. The level of the catalytic subunit of PKA decreases during aging, and acute activation of the cAMP/PKA pathway in aged flies strongly stimulates mitochondrial motility. Thus, the age-related impairment of transport is reversible. The expression of many genes is increased by PKA activation in aged flies. However, our results indicate that elevated mitochondrial transport is due in part to upregulation of the heavy chain of the kinesin-1 motor, the level of which declines during aging. Our study identifies evolutionarily conserved factors that can strongly influence mitochondrial motility in aging neurons. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.


    Devraj, Kavi; Klinger, Marianne E.; Myers, Roland L.; Mokashi, Ashwini; Hawkins, Richard A.; Simpson, Ian A.


    Glucose is the primary metabolic fuel for the mammalian brain and a continuous supply is required to maintain normal CNS function. The transport of glucose across the blood-brain barrier (BBB) into the brain is mediated by the facilitative glucose transporter GLUT-1. Prior studies (Simpson et al. 2001) had revealed that the conformations of the GLUT-1 transporter were different in luminal (blood facing) and abluminal (brain facing) membranes of bovine cerebral endothelial cells, based on differential antibody recognition. In this study we have extended these observations and using a combination of 2D-PAGE/Western blotting and immunogold electron microscopy we determined that these different conformations are exhibited in vivo and arise from differential phosphorylation of GLUT-1 and not from alternative splicing or altered O- or N-linked glycosylation. PMID:21910135

  13. Herpes Simplex Virus Membrane Proteins gE/gI and US9 Act Cooperatively To Promote Transport of Capsids and Glycoproteins from Neuron Cell Bodies into Initial Axon Segments (United States)

    Howard, Paul W.; Howard, Tiffani L.


    Herpes simplex virus (HSV) and other alphaherpesviruses must move from sites of latency in ganglia to peripheral epithelial cells. How HSV navigates in neuronal axons is not well understood. Two HSV membrane proteins, gE/gI and US9, are key to understanding the processes by which viral glycoproteins, unenveloped capsids, and enveloped virions are transported toward axon tips. Whether gE/gI and US9 function to promote the loading of viral proteins onto microtubule motors in neuron cell bodies or to tether viral proteins onto microtubule motors within axons is not clear. One impediment to understanding how HSV gE/gI and US9 function in axonal transport relates to observations that gE−, gI−, or US9− mutants are not absolutely blocked in axonal transport. Mutants are significantly reduced in numbers of capsids and glycoproteins in distal axons, but there are less extensive effects in proximal axons. We constructed HSV recombinants lacking both gE and US9 that transported no detectable capsids and glycoproteins to distal axons and failed to spread from axon tips to adjacent cells. Live-cell imaging of a gE−/US9− double mutant that expressed fluorescent capsids and gB demonstrated >90% diminished capsids and gB in medial axons and no evidence for decreased rates of transport, stalling, or increased retrograde transport. Instead, capsids, gB, and enveloped virions failed to enter proximal axons. We concluded that gE/gI and US9 function in neuron cell bodies, in a cooperative fashion, to promote the loading of HSV capsids and vesicles containing glycoproteins and enveloped virions onto microtubule motors or their transport into proximal axons. PMID:23077321

  14. The distribution of chandelier cell axon terminals that express the GABA plasma membrane transporter GAT-1 in the human neocortex. (United States)

    Inda, M C; Defelipe, J; Muñoz, A


    Chandelier cells represent a unique type of cortical GABAergic interneuron whose axon terminals (Ch-terminals) form synapses exclusively with the axon initial segments of pyramidal cells. In this study, we have used immunocytochemistry for the high-affinity plasma membrane transporter-1 (GAT-1) to analyze the distribution and density of Ch-terminals in various cytoarchitectonic and functional areas of the human neocortex. The lowest density of GAT-1-immuoreactive (-ir) Ch-terminals was detected in the primary and secondary visual (areas 17 and 18) and in the somatosensory areas (areas 3b and 1). In contrast, an intermediate density was observed in the motor area 4 and the associative frontolateral areas 45 and 46, whereas the associative frontolateral areas 9 and 10, frontal orbitary areas 11, 12, 13, 14, and 47, associative temporal areas 20, 21, 22, and 38, and cingulate areas 24 and 32 displayed the highest density of GAT-1-ir Ch-terminals. Despite these differences, the laminar distribution of GAT-1-ir Ch-terminals was similar in most cortical areas. Hence, the highest density of this transporter was observed in layer II, followed by layers III, V, VI, and IV. In most cortical areas, the density of GAT-1-ir Ch-terminals was positively correlated with the neuronal density, although a negative correlation was detected in layer III across all cortical areas. These results indicate that there are substantial differences in the distribution and density of GAT-1-ir Ch-terminals between areas and layers of the human neocortex. These differences might be related to the different functional attributes of the cortical regions examined.

  15. Phosphorylation of the norepinephrine transporter at threonine 258 and serine 259 is linked to protein kinase C-mediated transporter internalization

    DEFF Research Database (Denmark)

    Jayanthi, Lankupalle D; Annamalai, Balasubramaniam; Samuvel, Devadoss J


    ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation......Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested...

  16. MAP2 Defines a Pre-axonal Filtering Zone to Regulate KIF1- versus KIF5-Dependent Cargo Transport in Sensory Neurons

    NARCIS (Netherlands)

    Gumy, Laura F|info:eu-repo/dai/nl/337608334; Katrukha, Eugene A; Grigoriev, Ilya; Jaarsma, Dick; Kapitein, Lukas C|info:eu-repo/dai/nl/298806630; Akhmanova, Anna|info:eu-repo/dai/nl/156410591; Hoogenraad, Casper C|info:eu-repo/dai/nl/227263502


    Polarized cargo transport is essential for neuronal function. However, the minimal basic components required for selective cargo sorting and distribution in neurons remain elusive. We found that in sensory neurons the axon initial segment is largely absent and that microtubule-associated protein 2

  17. Nebula/DSCR1 upregulation delays neurodegeneration and protects against APP-induced axonal transport defects by restoring calcineurin and GSK-3β signaling.

    Directory of Open Access Journals (Sweden)

    Jillian L Shaw

    Full Text Available Post-mortem brains from Down syndrome (DS and Alzheimer's disease (AD patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1, but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, we explored the functional interaction between DSCR1 and the amyloid precursor protein (APP, which is known to cause AD when duplicated or upregulated in DS. We find that the Drosophila homolog of DSCR1, Nebula, delays neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, we show that Nebula upregulation protects against axonal transport defects by restoring calcineurin and GSK-3β signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, our findings imply that correcting calcineurin and GSK-3β signaling can prevent APP-induced pathologies. Our data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD.

  18. Restoration of active transport of solutes and oxidative phosphorylation by naphthoquinones in irradiated membrane vesicles from Mycobacterium phlei (United States)

    Lee, Soon-Ho; Sutherland, Thomas O.; Deves, Rosa; Brodie, Arnold F.


    Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK9(II-H)] and resulted in a loss of substrate oxidation, pH gradient, membrane potential, active transport of proline or calcium ions, and oxidative phosphorylation. Restoration of the protonmotive force and active transport occurred on addition of naphthoquinones such as vitamin K1, menadione, or lapachol to the irradiated membrane vesicles. However, coupled phosphorylation was restored only by vitamin K1. Menadione and lapachol did not act as uncoupling agents. The magnitude of the pH gradient and membrane potential in the quinone-restored system was a reflection of the rate of oxidation and was correlated with the rate of uptake of proline or Ca2+. These results are consistent with the chemosmotic hypothesis proposed for the energy transducing mechanism for active transport and further demonstrate that the complete respiratory chain is not required to drive active transport. In contrast, the data suggest that in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthaquinones, is essential for the utilization of the proton gradient or membrane potential or both. Bypass of electrons from the respiratory chain with menadione may explain the inability of this quinone to restore oxidative phosphorylation; however, lapachol restores oxidation by the same electron transport pathway as the natural menaquinone but fails to restore phosphorylation. Because all three quinones restore the protonmotive force, other factors that are discussed must be considered in understanding the mechanism of oxidative phosphorylation. PMID:6928606

  19. Lithium reverses behavioral and axonal transport-related changes associated with ANK3 bipolar disorder gene disruption. (United States)

    Gottschalk, Michael G; Leussis, Melanie P; Ruland, Tillmann; Gjeluci, Klaudio; Petryshen, Tracey L; Bahn, Sabine


    Ankyrin 3 (ANK3) has been implicated as a genetic risk factor for bipolar disorder (BD), however the resulting pathophysiological and treatment implications remain elusive. In a preclinical systems biological approach, we aimed to characterize the behavioral and proteomic effects of Ank3 haploinsufficiency and chronic mood-stabilizer treatment in mice. Psychiatric-related behavior was evaluated with the novelty-suppressed feeding (NSF) paradigm, elevated plus maze (EPM) and a passive avoidance task (PAT). Tandem mass spectrometry (MS E ) was employed for hippocampal proteome profiling. A functional enrichment approach based on protein-protein interactions (PPIs) was performed to outline which biological processes in the hippocampus were affected by Ank3 haploinsufficiency and lithium treatment. Proteomic abundance changes as detected by MS E or highlighted by PPI network modelling were followed up by targeted selected reaction monitoring (SRM). Increased psychiatric-related behavior in Ank3+/- mice was ameliorated by lithium in all assessments (NSF, EPM, PAT). MS E followed by modular PPI clustering and functional annotation enrichment pointed towards kinesin-related axonal transport and glutamate signaling as mediators of Ank3+/- pathophysiology and lithium treatment. SRM validated this hypothesis and further confirmed abundance changes of ANK3 interaction partners. We propose that psychiatric-related behavior in Ank3+/- mice is connected to a disturbance of the kinesin cargo system, resulting in a dysfunction of neuronal ion channel and glutamate receptor transport. Lithium reverses this molecular signature, suggesting the promotion of anterograde kinesin transport as part of its mechanism of action in ameliorating Ank3-related psychiatric-related behavior. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

  20. Organophosphate Related Alterations in Myelin and Axonal Transport in the Living Mammalian Brain (United States)


    standard 12-h light/dark cycle with free access to food (Teklad Global C.M. Hernandez et al. / NeuroToxicology 47 (2015) 17–26 19Rodent Diet 2918, Harlan...transport deficits in a triple transgenic mouse model of Alzheimer’s disease using manganese-enhanced MRI. Neuroimage 2011;56:1286–92. Lange G, Tiersky LA

  1. Herpes Simplex Virus gE/gI and US9 Promote both Envelopment and Sorting of Virus Particles in the Cytoplasm of Neurons, Two Processes That Precede Anterograde Transport in Axons. (United States)

    DuRaine, Grayson; Wisner, Todd W; Howard, Paul; Williams, Melissa; Johnson, David C


    Herpes simplex virus (HSV) anterograde transport in neuronal axons is vital, allowing spread from latently infected ganglia to epithelial tissues, where viral progeny are produced in numbers allowing spread to other hosts. The HSV membrane proteins gE/gI and US9 initiate the process of anterograde axonal transport, ensuring that virus particles are transported from the cytoplasm into the most proximal segments of axons. These proteins do not appear to be important once HSV is inside axons. We previously described HSV double mutants lacking both gE and US9 that failed to transport virus particles into axons. Here we show that gE - US9 - double mutants accumulate large quantities of unenveloped and partially enveloped capsids in neuronal cytoplasm. These defects in envelopment can explain the defects in axonal transport of enveloped virions. In addition, the unenveloped capsids that accumulated were frequently bound to cytoplasmic membranes, apparently immobilized in intermediate stages of envelopment. A gE-null mutant produced enveloped virions, but these accumulated in large numbers in the neuronal cytoplasm rather than reaching cell surfaces as wild-type HSV virions do. Thus, in addition to the defects in envelopment, there was missorting of capsids and enveloped particles in the neuronal cytoplasm, which can explain the reduced anterograde transport of unenveloped capsids and enveloped virions. These mechanisms differ substantially from existing models suggesting that gE/gI and US9 function by tethering HSV particles to kinesin microtubule motors. The defects in assembly of gE - US9 - mutant virus particles were novel because they were neuron specific, in keeping with observations that US9 is neuron specific. IMPORTANCE Herpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether themselves to dyneins and kinesins that motor along microtubules

  2. The axonal cytoskeleton : from organization to function

    NARCIS (Netherlands)

    Kevenaar, Josta T|info:eu-repo/dai/nl/338771042; Hoogenraad, Casper C|info:eu-repo/dai/nl/227263502

    The axon is the single long fiber that extends from the neuron and transmits electrical signals away from the cell body. The neuronal cytoskeleton, composed of microtubules (MTs), actin filaments and neurofilaments, is not only required for axon formation and axonal transport but also provides the

  3. Anterograde axonal transport and intercellular transfer of WGA-HRP in trigeminal-innervated sensory receptors of rat incisive papilla. (United States)

    Chan, K Y; Byers, M R


    The ultrastructure and identification of WGA-HRP-labeled sensory receptors in the rat incisive papilla (the most anterior part of hard palate) were studied using semiserial thin sections. Various sensory receptors were organized according to three locations: dome region (ventral), chemosensory corpuscle region (medial to orifice of incisive canal), and lateral labium (apposing the incisive canal). In the dome region, the sensory receptors were localized in three sensory zones that were associated with surface ridges (one medial and two lateral). In each of these zones, intraepithelial receptor axons and Merkel receptors occurred in the epithelium, while simple unencapsulated corpuscles, glomerular-Meissner corpuscles, and incisive (encapsulated) corpuscles occurred in the lamina propria. In the chemosensory corpuscle region, chemosensory corpuscles and intraepithelial receptor axons were located in the epithelium, and incisive corpuscles were present in the lamina propria. In the lateral labium, only intraepithelial receptor axons were prominent. In all these sensory receptors, the preterminal axons and axon terminals were labeled with the tracer protein. In addition, some nonneuronal cells closely associated with the axon terminals were selectively labeled, e.g., terminal Schwann cells, lamellar Schwann cells, Merkel cells, corpuscular basal cells and chemosensory cells. Other adjacent cells were not labeled, e.g., unspecialized epithelial cells, capsular cells, corpuscular sustentacular cells, and fibroblasts. In both labeled axons and cells, WGA-HRP was incorporated into vesicles, tubules, and vacuolar organelles. The specific intercellular transfer of tracer protein may indicate trophic interactions between axon terminals and support cells in sensory receptors. The specific organization of multiple sensory receptors in the rat incisive papilla may provide a useful alternative system for studying somatosensory physiology.

  4. Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

    Energy Technology Data Exchange (ETDEWEB)

    Bligny, R.; Foray, M.F.; Roby, C.; Douce, R.


    When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by /sup 31/P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.

  5. N-terminal truncation of the dopamine transporter abolishes phorbol ester- and substance P receptor-stimulated phosphorylation without impairing transporter internalization

    DEFF Research Database (Denmark)

    Granas, Charlotta; Ferrer, Jasmine; Loland, Claus Juul


    The structural basis of phosphorylation and its putative role in internalization were investigated in the human dopamine transporter (hDAT). Activation of protein kinase C (PKC) was achieved either directly by treatment with 4-alpha-phorbol 12-myristate 13-acetate (PMA) or by activating the Galpha...

  6. Sorting of Dendritic and Axonal Vesicles at the Pre-axonal Exclusion Zone

    Directory of Open Access Journals (Sweden)

    Ginny G. Farías


    Full Text Available Polarized sorting of newly synthesized proteins to the somatodendritic and axonal domains of neurons occurs by selective incorporation into distinct populations of vesicular transport carriers. An unresolved issue is how the vesicles themselves are sorted to their corresponding neuronal domains. Previous studies concluded that the axon initial segment (AIS is an actin-based filter that selectively prevents passage of somatodendritic vesicles into the axon. We find, however, that most somatodendritic vesicles fail to enter the axon at a more proximal region in the axon hillock, herein referred to as the pre-axonal exclusion zone (PAEZ. Forced coupling of a somatodendritic cargo protein to an axonally directed kinesin is sufficient to drive transport of whole somatodendritic vesicles through the PAEZ toward the distal axon. Based on these findings, we propose that polarized sorting of transport vesicles occurs at the PAEZ and depends on the ability of the vesicles to acquire an appropriately directed microtubule motor.

  7. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

    Energy Technology Data Exchange (ETDEWEB)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik


    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  8. Reduced coupling of oxidative phosphorylation in vivo precedes electron transport chain defects due to mild oxidative stress in mice.

    Directory of Open Access Journals (Sweden)

    Michael P Siegel

    Full Text Available Oxidative stress and mitochondrial function are at the core of many degenerative conditions. However, the interaction between oxidative stress and in vivo mitochondrial function is unclear. We used both pharmacological (2 week paraquat (PQ treatment of wild type mice and transgenic (mice lacking Cu, Zn-superoxide dismutase (SOD1(-/- models to test the effect of oxidative stress on in vivo mitochondrial function in skeletal muscle. Magnetic resonance and optical spectroscopy were used to measure mitochondrial ATP and oxygen fluxes and cell energetic state. In both models of oxidative stress, coupling of oxidative phosphorylation was significantly lower (lower P/O at rest in vivo in skeletal muscle and was dose-dependent in the PQ model. Despite this reduction in efficiency, in vivo mitochondrial phosphorylation capacity (ATPmax was maintained in both models, and ex vivo mitochondrial respiration in permeabilized muscle fibers was unchanged following PQ treatment. In association with the reduced P/O, PQ treatment led to a dose-dependent reduction in PCr/ATP ratio and increased phosphorylation of AMPK. These results indicate that oxidative stress uncouples oxidative phosphorylation in vivo and results in energetic stress in the absence of defects in the mitochondrial electron transport chain.

  9. PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress. (United States)

    Huang, En; Qu, Dianbo; Huang, Tianwen; Rizzi, Nicoletta; Boonying, Wassamon; Krolak, Dorothy; Ciana, Paolo; Woulfe, John; Klein, Christine; Slack, Ruth S; Figeys, Daniel; Park, David S


    Mutations in PTEN-induced kinase 1 (PINK1) result in a recessive familial form of Parkinson's disease (PD). PINK1 loss is associated with mitochondrial Ca 2+ mishandling, mitochondrial dysfunction, as well as increased neuronal vulnerability. Here we demonstrate that PINK1 directly interacts with and phosphorylates LETM1 at Thr192 in vitro. Phosphorylated LETM1 or the phospho-mimetic LETM1-T192E increase calcium release in artificial liposomes and facilitates calcium transport in intact mitochondria. Expression of LETM1-T192E but not LETM1-wild type (WT) rescues mitochondrial calcium mishandling in PINK1-deficient neurons. Expression of both LETM1-WT and LETM1-T192E protects neurons against MPP + -MPTP-induced neuronal death in PINK1 WT neurons, whereas only LETM1-T192E protects neurons under conditions of PINK1 loss. Our findings delineate a mechanism by which PINK1 regulates mitochondrial Ca 2+ level through LETM1 and suggest a model by which PINK1 loss leads to deficient phosphorylation of LETM1 and impaired mitochondrial Ca 2+ transport..

  10. Spastic paraplegia mutation N256S in the neuronal microtubule motor KIF5A disrupts axonal transport in a Drosophila HSP model.

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    Petra Füger

    Full Text Available Hereditary spastic paraplegias (HSPs comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10, caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP-like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.

  11. Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons

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    Katherina Beck


    Full Text Available Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2 acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.

  12. Cell intrinsic control of axon regeneration (United States)

    Mar, Fernando M; Bonni, Azad; Sousa, Mónica M


    Although neurons execute a cell intrinsic program of axonal growth during development, following the establishment of connections, the developmental growth capacity declines. Besides environmental challenges, this switch largely accounts for the failure of adult central nervous system (CNS) axons to regenerate. Here, we discuss the cell intrinsic control of axon regeneration, including not only the regulation of transcriptional and epigenetic mechanisms, but also the modulation of local protein translation, retrograde and anterograde axonal transport, and microtubule dynamics. We further explore the causes underlying the failure of CNS neurons to mount a vigorous regenerative response, and the paradigms demonstrating the activation of cell intrinsic axon growth programs. Finally, we present potential mechanisms to support axon regeneration, as these may represent future therapeutic approaches to promote recovery following CNS injury and disease. PMID:24531721

  13. Novel vasopressin-regulated phosphorylation site of the NaCl co-transporter, NCC

    DEFF Research Database (Denmark)

    Rosenbæk, Lena Lindtoft; Knepper, Mark A; Fenton, Robert A.

    with a novel pS124-NCC antibody demonstrated a band of ~160 kDa in the kidney cortex, but not medulla, which was preabsorbed by a corresponding phosphorylated peptide. Confocal microscopy with segment-specific markers localized pS124-NCC to all DCT cells, with greater abundance in the early DCT. Double...... immunogold electron microscopy with total NCC revealed that pS124-NCC was associated predominantly with the apical plasma membrane of DCT cells, although some labelling was associated with intracellular vesicles. Acute, but not long-term, vasopressin treatment significantly increased pS124-NCC abundance...... at the plasma membrane. Similar observations were apparent after rats were fed a low salt diet. Kinase profiling assays using a synthetic peptide corresponding to the region surrounding S124 against the protein kinases mTOR/FRAP, ERK5, NLK, p38d, and ERK2 showed either weak or no significant activity. Funded...

  14. Axonal interferon responses and alphaherpesvirus neuroinvasion (United States)

    Song, Ren

    Infection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at a peripheral epithelial surface and continues into the peripheral nervous system (PNS) that innervates this tissue. Inflammatory responses are induced at the infected peripheral site prior to viral invasion of the PNS. PNS neurons are highly polarized cells with long axonal processes that connect to distant targets. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which include type I interferon (e.g. IFNbeta) and type II interferon (i.e. IFNgamma). IFNbeta can be produced by all types of cells, while IFNgamma is secreted by some specific types of immune cells. And both types of IFN induce antiviral responses in surrounding cells that express the IFN receptors. The fundamental question is how do PNS neurons respond to the inflammatory milieu experienced only by their axons. Axons must act as potential front-line barriers to prevent PNS infection and damage. Using compartmented cultures that physically separate neuron axons from cell bodies, I found that pretreating isolated axons with IFNbeta or IFNgamma significantly diminished the number of HSV-1 and PRV particles moving from axons to the cell bodies in an IFN receptor-dependent manner. Furthermore, I found the responses in axons are activated differentially by the two types of IFNs. The response to IFNbeta is a rapid, axon-only response, while the response to IFNgamma involves long distance signaling to the PNS cell body. For example, exposing axons to IFNbeta induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFNgamma induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated IFNgamma-, but not IFNbeta-mediated antiviral effects. Proteomic analysis of IFNbeta- or IFNgamma-treated axons identified several differentially regulated proteins. Therefore

  15. Measurement of glucose and 2-deoxy-2-[18F]fluoro-D-glucose transport and phosphorylation rates in myocardium using dual-tracer kinetic experiments

    International Nuclear Information System (INIS)

    Huang, S.C.; Williams, B.A.; Barrio, J.R.; Nissenson, C.; Hoffman, E.J.; Phelps, M.E.; Krivokapich, J.


    To examine the use of 2-deoxy-2-[ 18 F]fluoro-D-glucose (2-FDG) as a glucose analog for measuring glucose utilization rate in myocardium, dual-tracer kinetic experiments with 2-FDG and 2-[ 3 H]glucose were performed in the perfused, isolated rabbit interventricular septum to measure simultaneously the transport and phosphorylation rates of glucose and 2-FDG. Results of the present study indicated that, in the septum, (i) the transport rate constants of 2-FDG and glucose were similar in magnitude, (ii) the phosphorylation rate constant for 2-FDG was about 60% of that of glucose, (iii) hypoxia caused an increase in phosphorylation rates of glucose and 2-FDG without affecting transport. 9 refs.; 1 figure; 3 tabs

  16. The C-terminal domains of NF-H and NF-M subunits maintain axonal neurofilament content by blocking turnover of the stationary neurofilament network.

    Directory of Open Access Journals (Sweden)

    Mala V Rao

    Full Text Available Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M(tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M(tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M(tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.

  17. Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating

    DEFF Research Database (Denmark)

    Moeller, Hanne B; Macaulay, Nanna; Knepper, Mark A


    demonstrated that lack of phosphorylation at S256, S261, S264, or S269 had no effect on AQP2 unit water transport. Similarly, no effect on AQP2 unit water transport was observed for the 264D and 269D forms, indicating that phosphorylation of the COOH-terminal tail of AQP2 is not involved in gating......Arginine vasopressin (AVP)-regulated phosphorylation of the water channel aquaporin-2 (AQP2) at serine 256 (S256) is essential for its accumulation in the apical plasma membrane of collecting duct principal cells. In this study, we examined the role of additional AVP-regulated phosphorylation sites...... in the COOH-terminal tail of AQP2 on protein function. When expressed in Xenopus laevis oocytes, prevention of AQP2 phosphorylation at S256A (S256A-AQP2) reduced osmotic water permeability threefold compared with wild-type (WT) AQP2-injected oocytes. In contrast, prevention of AQP2 single phosphorylation at S...

  18. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility (United States)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.


    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  19. Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons. (United States)

    Beck, Katherina; Ehmann, Nadine; Andlauer, Till F M; Ljaschenko, Dmitrij; Strecker, Katrin; Fischer, Matthias; Kittel, Robert J; Raabe, Thomas


    Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling. © 2015. Published by The Company of Biologists Ltd.

  20. Active Transport of Phosphorylated Carbohydrates Promotes Intestinal Colonization and Transmission of a Bacterial Pathogen.

    Directory of Open Access Journals (Sweden)

    Brandon Sit


    Full Text Available Efficient acquisition of extracellular nutrients is essential for bacterial pathogenesis, however the identities and mechanisms for transport of many of these substrates remain unclear. Here, we investigate the predicted iron-binding transporter AfuABC and its role in bacterial pathogenesis in vivo. By crystallographic, biophysical and in vivo approaches, we show that AfuABC is in fact a cyclic hexose/heptose-phosphate transporter with high selectivity and specificity for a set of ubiquitous metabolites (glucose-6-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate. AfuABC is conserved across a wide range of bacterial genera, including the enteric pathogens EHEC O157:H7 and its murine-specific relative Citrobacter rodentium, where it lies adjacent to genes implicated in sugar sensing and acquisition. C. rodentium ΔafuA was significantly impaired in an in vivo murine competitive assay as well as its ability to transmit infection from an afflicted to a naïve murine host. Sugar-phosphates were present in normal and infected intestinal mucus and stool samples, indicating that these metabolites are available within the intestinal lumen for enteric bacteria to import during infection. Our study shows that AfuABC-dependent uptake of sugar-phosphates plays a critical role during enteric bacterial infection and uncovers previously unrecognized roles for these metabolites as important contributors to successful pathogenesis.

  1. HPr(His~P)-mediated Phosphorylation Differently Affects Counterflow and Proton Motive Force-driven Uptake via the Lactose Transport Protein of Streptococcus thermophilus

    NARCIS (Netherlands)

    Gunnewijk, M.G W; Poolman, B.


    The lactose transport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The IIA domain of LacS is phosphorylated on His-552 by the general

  2. Phosphorylation states of the (Na+ + K+)-transporting ATPase in preparations from lamb kidney and electric-eel (Electophorus electricus) electric organ. (United States)

    Harris, W E; Stahl, W L


    Phosphorylation states of the (Na+ + K+)-transporting ATPase were studied in highly purified preparations isolated from electric-eel electric organ and from lamb kidney. The steady-state level of phosphorylated lamb kidney enzyme, obtained by reaction with [gamma-32P]ATP, was not appreciably reduced in the presence of ADP unless oligomycin was present. The phosphorylated form of the electric-eel electric-organ enzyme was reduced by at least 95% under the same conditions, suggesting that the E1P state in the kidney enzyme is more transitory than that in electric organ. The level of phosphorylation from [32P]Pi was higher in the lamb kidney preparation than in the electric-organ preparation, and the difference in stimulation of phosphorylation by ouabain in the two preparations was striking. Ouabain increased the level of phosphorylation by 35% in the kidney preparation and 734% in the electric-organ preparation. The E2P state seems to be stabilized by ouabain in the latter preparation. These findings, as well as the different reactivities of the thiol groups to blocking reagents in these preparations, suggest that the tertiary structure in the enzyme isolated from these two sources is different. PMID:6324756

  3. Torsional Behavior of Axonal Microtubule Bundles (United States)

    Lazarus, Carole; Soheilypour, Mohammad; Mofrad, Mohammad R.K.


    Axonal microtubule (MT) bundles crosslinked by microtubule-associated protein (MAP) tau are responsible for vital biological functions such as maintaining mechanical integrity and shape of the axon as well as facilitating axonal transport. Breaking and twisting of MTs have been previously observed in damaged undulated axons. Such breaking and twisting of MTs is suggested to cause axonal swellings that lead to axonal degeneration, which is known as “diffuse axonal injury”. In particular, overstretching and torsion of axons can potentially damage the axonal cytoskeleton. Following our previous studies on mechanical response of axonal MT bundles under uniaxial tension and compression, this work seeks to characterize the mechanical behavior of MT bundles under pure torsion as well as a combination of torsional and tensile loads using a coarse-grained computational model. In the case of pure torsion, a competition between MAP tau tensile and MT bending energies is observed. After three turns, a transition occurs in the mechanical behavior of the bundle that is characterized by its diameter shrinkage. Furthermore, crosslink spacing is shown to considerably influence the mechanical response, with larger MAP tau spacing resulting in a higher rate of turns. Therefore, MAP tau crosslinking of MT filaments protects the bundle from excessive deformation. Simultaneous application of torsion and tension on MT bundles is shown to accelerate bundle failure, compared to pure tension experiments. MAP tau proteins fail in clusters of 10–100 elements located at the discontinuities or the ends of MT filaments. This failure occurs in a stepwise fashion, implying gradual accumulation of elastic tensile energy in crosslinks followed by rupture. Failure of large groups of interconnecting MAP tau proteins leads to detachment of MT filaments from the bundle near discontinuities. This study highlights the importance of torsional loading in axonal damage after traumatic brain injury

  4. Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells


    Klein, Janet D.; Blount, Mitsi A.; Fröhlich, Otto; Denson, Chad E.; Tan, Xiaoxiao; Sim, Jae H.; Martin, Christopher F.; Sands, Jeff M.


    Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-A1 urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-A1 phosphorylation at S486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed S486 in roughly the center of the sequence. We also developed two stably transfected mIMCD3 cell lines: one expressing wild-ty...

  5. Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation

    DEFF Research Database (Denmark)

    Henrichs, Sina; Wang, Bangjun; Fukao, Yoichiro


    Polar transport of the plant hormone auxin is controlled by PIN-and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co......-IP) and shotgun LC-MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. In-vitro and yeast expression analyses indicated that PID specifically modulates ABCB1-mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding...... and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co-transfection in tobacco revealed that PID enhances ABCB1-mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho-proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely...

  6. Diffuse Axonal Injury and Oxidative Stress: A Comprehensive Review

    Directory of Open Access Journals (Sweden)

    Alessandro Frati


    Full Text Available Traumatic brain injury (TBI is one of the world’s leading causes of morbidity and mortality among young individuals. TBI applies powerful rotational and translational forces to the brain parenchyma, which results in a traumatic diffuse axonal injury (DAI responsible for brain swelling and neuronal death. Following TBI, axonal degeneration has been identified as a progressive process that starts with disrupted axonal transport causing axonal swelling, followed by secondary axonal disconnection and Wallerian degeneration. These modifications in the axonal cytoskeleton interrupt the axoplasmic transport mechanisms, causing the gradual gathering of transport products so as to generate axonal swellings and modifications in neuronal homeostasis. Oxidative stress with consequent impairment of endogenous antioxidant defense mechanisms plays a significant role in the secondary events leading to neuronal death. Studies support the role of an altered axonal calcium homeostasis as a mechanism in the secondary damage of axon, and suggest that calcium channel blocker can alleviate the secondary damage, as well as other mechanisms implied in the secondary injury, and could be targeted as a candidate for therapeutic approaches. Reactive oxygen species (ROS-mediated axonal degeneration is mainly caused by extracellular Ca2+. Increases in the defense mechanisms through the use of exogenous antioxidants may be neuroprotective, particularly if they are given within the neuroprotective time window. A promising potential therapeutic target for DAI is to directly address mitochondria-related injury or to modulate energetic axonal energy failure.

  7. Diffuse Axonal Injury and Oxidative Stress: A Comprehensive Review. (United States)

    Frati, Alessandro; Cerretani, Daniela; Fiaschi, Anna Ida; Frati, Paola; Gatto, Vittorio; La Russa, Raffaele; Pesce, Alessandro; Pinchi, Enrica; Santurro, Alessandro; Fraschetti, Flavia; Fineschi, Vittorio


    Traumatic brain injury (TBI) is one of the world's leading causes of morbidity and mortality among young individuals. TBI applies powerful rotational and translational forces to the brain parenchyma, which results in a traumatic diffuse axonal injury (DAI) responsible for brain swelling and neuronal death. Following TBI, axonal degeneration has been identified as a progressive process that starts with disrupted axonal transport causing axonal swelling, followed by secondary axonal disconnection and Wallerian degeneration. These modifications in the axonal cytoskeleton interrupt the axoplasmic transport mechanisms, causing the gradual gathering of transport products so as to generate axonal swellings and modifications in neuronal homeostasis. Oxidative stress with consequent impairment of endogenous antioxidant defense mechanisms plays a significant role in the secondary events leading to neuronal death. Studies support the role of an altered axonal calcium homeostasis as a mechanism in the secondary damage of axon, and suggest that calcium channel blocker can alleviate the secondary damage, as well as other mechanisms implied in the secondary injury, and could be targeted as a candidate for therapeutic approaches. Reactive oxygen species (ROS)-mediated axonal degeneration is mainly caused by extracellular Ca 2+ . Increases in the defense mechanisms through the use of exogenous antioxidants may be neuroprotective, particularly if they are given within the neuroprotective time window. A promising potential therapeutic target for DAI is to directly address mitochondria-related injury or to modulate energetic axonal energy failure.

  8. Dync1h1 Mutation Causes Proprioceptive Sensory Neuron Loss and Impaired Retrograde Axonal Transport of Dorsal Root Ganglion Neurons. (United States)

    Zhao, Jing; Wang, Yi; Xu, Huan; Fu, Yuan; Qian, Ting; Bo, Deng; Lu, Yan-Xin; Xiong, Yi; Wan, Jun; Zhang, Xiang; Dong, Qiang; Chen, Xiang-Jun


    Sprawling (Swl) is a radiation-induced mutation which has been identified to have a nine base pair deletion in dynein heavy chain 1 (DYNC1H1: encoded by a single gene Dync1h1). This study is to investigate the phenotype and the underlying mechanism of the Dync1h1 mutant. To display the phenotype of Swl mutant mice, we examined the embryos of homozygous (Swl/Swl) and heterozygous (Swl/+) mice and their postnatal dorsal root ganglion (DRG) of surviving Swl/+ mice. The Swl/+ mice could survive for a normal life span, while Swl/Swl could only survive till embryonic (E) 8.5 days. Excessive apoptosis of Swl/+ DRG neurons was revealed during E11.5-E15.5 days, and the peak rate was at E13.5 days. In vitro study of mutated DRG neurons showed impaired retrograde transport of dynein-driven nerve growth factor (NGF). Mitochondria, another dynein-driven cargo, demonstrated much slower retrograde transport velocity in Swl/+ neurons than in wild-type (WT) neurons. Nevertheless, the Swl, Loa, and Cra mutations did not affect homodimerization of DYNC1H1. The Swl/Swl mutation of Dync1h1 gene led to embryonic mal-development and lethality, whereas the Swl/+ DRG neurons demonstrated deficient retrograde transport in dynein-driven cargos and excessive apoptosis during mid- to late-developmental stages. The underlying mechanism of the mutation may not be due to impaired homodimerization of DYNC1H1. © 2016 John Wiley & Sons Ltd.

  9. Defective lysosomal proteolysis and axonal transport are early pathogenic events that worsen with age leading to increased APP metabolism and synaptic Abeta in transgenic APP/PS1 hippocampus

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    Torres Manuel


    Full Text Available Abstract Background Axonal pathology might constitute one of the earliest manifestations of Alzheimer disease. Axonal dystrophies were observed in Alzheimer’s patients and transgenic models at early ages. These axonal dystrophies could reflect the disruption of axonal transport and the accumulation of multiple vesicles at local points. It has been also proposed that dystrophies might interfere with normal intracellular proteolysis. In this work, we have investigated the progression of the hippocampal pathology and the possible implication in Abeta production in young (6 months and aged (18 months PS1(M146L/APP(751sl transgenic mice. Results Our data demonstrated the existence of a progressive, age-dependent, formation of axonal dystrophies, mainly located in contact with congophilic Abeta deposition, which exhibited tau and neurofilament hyperphosphorylation. This progressive pathology was paralleled with decreased expression of the motor proteins kinesin and dynein. Furthermore, we also observed an early decrease in the activity of cathepsins B and D, progressing to a deep inhibition of these lysosomal proteases at late ages. This lysosomal impairment could be responsible for the accumulation of LC3-II and ubiquitinated proteins within axonal dystrophies. We have also investigated the repercussion of these deficiencies on the APP metabolism. Our data demonstrated the existence of an increase in the amyloidogenic pathway, which was reflected by the accumulation of hAPPfl, C99 fragment, intracellular Abeta in parallel with an increase in BACE and gamma-secretase activities. In vitro experiments, using APPswe transfected N2a cells, demonstrated that any imbalance on the proteolytic systems reproduced the in vivo alterations in APP metabolism. Finally, our data also demonstrated that Abeta peptides were preferentially accumulated in isolated synaptosomes. Conclusion A progressive age-dependent cytoskeletal pathology along with a reduction of

  10. Deletion mutants of the Escherichia coli K-12 mannitol permease: dissection of transport-phosphorylation, phospho-exchange, and mannitol-binding activities. (United States)

    Grisafi, P L; Scholle, A; Sugiyama, J; Briggs, C; Jacobson, G R; Lengeler, J W


    We have constructed a series of deletion mutations of the cloned Escherichia coli K-12 mtlA gene, which encodes the mannitol-specific enzyme II of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system. This membrane-bound permease consists of 637 amino acid residues and is responsible for the concomitant transport and phosphorylation of D-mannitol in E. coli. Deletions into the 3' end of mtlA were constructed by exonuclease III digestion. Restriction mapping of the resultant plasmids identified several classes of deletions that lacked approximately 5% to more than 75% of the gene. Immunoblotting experiments revealed that many of these plasmids expressed proteins within the size range predicted by the restriction analyses, and all of these proteins were membrane localized, which demonstrated that none of the C-terminal half of the permease is required for membrane insertion. Functional analyses of the deletion proteins, expressed in an E. coli strain deleted for the chromosomal copy of mtlA, showed that all but one of the strains containing confirmed deletions were inactive in transport and PEP-dependent phosphorylation of mannitol, but deletions removing up to at least 117 amino acid residues from the C terminus of the permease were still active in catalyzing phospho exchange between mannitol 1-phosphate and mannitol. A deletion protein that lacked 240 residues from the C terminus of the permease was inactive in phospho exchange but still bound mannitol with high affinity. These experiments localize sites important for transport and PEP-dependent phosphorylation to the extreme C terminus of the mannitol permease, sites important for phospho exchange to between residues 377 and 519, and sites necessary for mannitol binding to the N-terminal 60% of the molecule. The results are discussed with respect to the fact that the mannitol permease consists of structurally independent N- and C-terminal domains.

  11. Can injured adult CNS axons regenerate by recapitulating development? (United States)

    Hilton, Brett J; Bradke, Frank


    In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS. © 2017. Published by The Company of Biologists Ltd.

  12. Acute nutritional axonal neuropathy. (United States)

    Hamel, Johanna; Logigian, Eric L


    This study describes clinical, laboratory, and electrodiagnostic features of a severe acute axonal polyneuropathy common to patients with acute nutritional deficiency in the setting of alcoholism, bariatric surgery (BS), or anorexia. Retrospective analysis of clinical, electrodiagnostic, and laboratory data of patients with acute axonal neuropathy. Thirteen patients were identified with a severe, painful, sensory or sensorimotor axonal polyneuropathy that developed over 2-12 weeks with sensory ataxia, areflexia, variable muscle weakness, poor nutritional status, and weight loss, often with prolonged vomiting and normal cerebrospinal fluid protein. Vitamin B6 was low in half and thiamine was low in all patients when obtained before supplementation. Patients improved with weight gain and vitamin supplementation, with motor greater than sensory recovery. We suggest that acute or subacute axonal neuropathy in patients with weight loss or vomiting associated with alcohol abuse, BS, or dietary deficiency is one syndrome, caused by micronutrient deficiencies. Muscle Nerve 57: 33-39, 2018. © 2017 Wiley Periodicals, Inc.

  13. Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells

    International Nuclear Information System (INIS)

    Imamura, K.; Schulz, I.


    Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca 2+ + Mg 2+ )-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32 P from [gamma- 32 P]ATP is Ca 2+ -dependent (approximate Km for free [Ca 2+ ] = 2-3 x 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32 P incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg 2+ and monovalent cations such as K + or Na + are necessary. Vanadate inhibits both 32 P incorporation and 32 P liberation dose dependently (Km = 3 x 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32 P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca 2+ , cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca 2+ transport into rough endoplasmic reticulum from pancreatic acinar cells. The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase

  14. Concepts for regulation of axon integrity by enwrapping glia

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    Bogdan eBeirowski


    Full Text Available Long axons and their enwrapping glia (Schwann cells and oligodendrocytes form a unique compound structure that serves as conduit for transport of electric and chemical information in the nervous system. The peculiar cytoarchitecture over an enormous length as well as its substantial energetic requirements make this conduit particularly susceptible to detrimental alterations. Degeneration of long axons independent of neuronal cell bodies is observed comparatively early in a range of neurodegenerative conditions as a consequence of abnormalities in Schwann cells and oligodendrocytes. This leads to the most relevant disease symptoms and highlights the critical role that these glia have for axon integrity, but the underlying mechanisms remain elusive. The quest to understand why and how axons degenerate is now a crucial frontier in disease-oriented research. This challenge is most likely to lead to significant progress if the inextricable link between axons and their flanking glia in pathological situations is recognized. In this review I compile recent advances in our understanding of the molecular programs governing axon degeneration, and mechanisms of enwrapping glia’s non-cell autonomous impact on axon-integrity. A particular focus is placed on emerging evidence suggesting that enwrapping glia nurture long axons by virtue of their intimate association, release of trophic substances, and neurometabolic coupling. The correction of defects in these functions has the potential to stabilize axons in a variety of neuronal diseases in the peripheral and central nervous system.

  15. Protein-synthesizing machinery in the axon compartment. (United States)

    Koenig, E; Giuditta, A


    Contrary to the prevailing view that the axon lacks the capacity to synthesize proteins, a substantial body of evidence points to the existence of a metabolically active endogenous translational machinery. The machinery appears to be largely localized in the cortical zone of the axon, where, in vertebrate axons, it is distributed longitudinally as intermittent, discrete domains, called periaxoplasmic plaques. Studies, based on translation assays and probes of RNA transcripts in axon models such as the squid giant axon and selected vertebrate axons, provide evidence of locally synthesized proteins, most of which appear to be constituents of the slow axoplasmic transport rate groups. Metabolic and molecular biological findings are consistent with the view that the synthesis of proteins undergoing local turnover in the axonal compartment of macroneurons depends on the activity of an endogenous translational machinery. The documented presence of a metabolically active machinery in presynaptic terminals of squid photoreceptor neurons is also described. Finally, potential sources of axoplasmic RNAs comprising the machinery, which may include the ensheathing cell of the axon, as well as the cognate cell body, are also discussed.

  16. Mitotic motors coregulate microtubule patterns in axons and dendrites. (United States)

    Lin, Shen; Liu, Mei; Mozgova, Olga I; Yu, Wenqian; Baas, Peter W


    Microtubules are nearly uniformly oriented in the axons of vertebrate neurons but are non-uniformly oriented in their dendrites. Studies to date suggest a scenario for establishing these microtubule patterns whereby microtubules are transported into the axon and nascent dendrites with plus-ends-leading, and then additional microtubules of the opposite orientation are transported into the developing dendrites. Here, we used contemporary tools to confirm that depletion of kinesin-6 (also called CHO1/MKLP1 or kif23) from rat sympathetic neurons causes a reduction in the appearance of minus-end-distal microtubules in developing dendrites, which in turn causes them to assume an axon-like morphology. Interestingly, we observed a similar phenomenon when we depleted kinesin-12 (also called kif15 or HKLP2). Both motors are best known for their participation in mitosis in other cell types, and both are enriched in the cell body and dendrites of neurons. Unlike kinesin-12, which is present throughout the neuron, kinesin-6 is barely detectable in the axon. Accordingly, depletion of kinesin-6, unlike depletion of kinesin-12, has no effect on axonal branching or navigation. Interestingly, depletion of either motor results in faster growing axons with greater numbers of mobile microtubules. Based on these observations, we posit a model whereby these two motors generate forces that attenuate the transport of microtubules with plus-ends-leading from the cell body into the axon. Some of these microtubules are not only prevented from moving into the axon but are driven with minus-ends-leading into developing dendrites. In this manner, these so-called "mitotic" motors coregulate the microtubule patterns of axons and dendrites.

  17. Signal propagation along the axon. (United States)

    Rama, Sylvain; Zbili, Mickaël; Debanne, Dominique


    Axons link distant brain regions and are usually considered as simple transmission cables in which reliable propagation occurs once an action potential has been generated. Safe propagation of action potentials relies on specific ion channel expression at strategic points of the axon such as nodes of Ranvier or axonal branch points. However, while action potentials are generally considered as the quantum of neuronal information, their signaling is not entirely digital. In fact, both their shape and their conduction speed have been shown to be modulated by activity, leading to regulations of synaptic latency and synaptic strength. We report here newly identified mechanisms of (1) safe spike propagation along the axon, (2) compartmentalization of action potential shape in the axon, (3) analog modulation of spike-evoked synaptic transmission and (4) alteration in conduction time after persistent regulation of axon morphology in central neurons. We discuss the contribution of these regulations in information processing. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Axonal and Transynaptic Spread of Prions (United States)

    Shearin, Harold


    ABSTRACT Natural transmission of prion diseases depends upon the spread of prions from the nervous system to excretory or secretory tissues, but the mechanism of prion transport in axons and into peripheral tissue is unresolved. Here, we examined the temporal and spatial movement of prions from the brain stem along cranial nerves into skeletal muscle as a model of axonal transport and transynaptic spread. The disease-specific isoform of the prion protein, PrPSc, was observed in nerve fibers of the tongue approximately 2 weeks prior to PrPSc deposition in skeletal muscle. Initially, PrPSc deposits had a small punctate pattern on the edge of muscle cells that colocalized with synaptophysin, a marker for the neuromuscular junction (NMJ), in >50% of the cells. At later time points PrPSc was widely distributed in muscle cells, but PrPSc deposition at the NMJ, suggesting additional prion replication and dissemination within muscle cells. In contrast to the NMJ, PrPSc was not associated with synaptophysin in nerve fibers but was found to colocalize with LAMP-1 and cathepsin D during early stages of axonal spread. We propose that PrPSc-bound endosomes can lead to membrane recycling in which PrPSc is directed to the synapse, where it either moves across the NMJ into the postsynaptic muscle cell or induces PrPSc formation on muscle cells across the NMJ. IMPORTANCE Prion diseases are transmissible and fatal neurodegenerative diseases in which prion dissemination to excretory or secretory tissues is necessary for natural disease transmission. Despite the importance of this pathway, the cellular mechanism of prion transport in axons and into peripheral tissue is unresolved. This study demonstrates anterograde spread of prions within nerve fibers prior to infection of peripheral synapses (i.e., neuromuscular junction) and infection of peripheral tissues (i.e., muscle cells). Within nerve fibers prions were associated with the endosomal-lysosomal pathway prior to entry into

  19. [The comparative characteristics of the age-related changes in the rate of fast axonal transport in the vagus and hypoglossal nerves and in the ventral spinal cord roots of rats]. (United States)

    Tanin, S A; Martsinko, V I


    The study was undertaken on the fast axonal transport (FAT) of 3H- or 14C-leucine labelled substances along the n. vagus, n. hypoglossus and ventral roots of the spinal cord in adult (8-10 months) and old (26-28 months) male rats after the label administration into nucleus ambiguus, nucleus hypoglossus, and the area of the ventral horn of the spinal cord, respectively. It has been found that in old rats compared to adult animals the rate of FAT along the n. vagus decreased from 552 +/- 12.7 mm to 252 +/- 13 mm per 24 hours; along the n. hypoglossus--from 492 +/- 38 mm to 216 mm per 24 hours; and along the ventral L5 and L6 roots--from 408 +/- 10.9 mm to 217 +/- 11.3 mm per 24 hours. It is suggested that age-related functional shifts in n. vagus influencing the heart are, to some degree, determined by the most significant disturbances of FAT substances in it.

  20. Axon-glia interaction and membrane traffic in myelin formation

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    Robin eWhite


    Full Text Available In vertebrate nervous systems myelination of neuronal axons has evolved to increase conduction velocity of electrical impulses with minimal space and energy requirements. Myelin is formed by specialised glial cells which ensheath axons with a lipid-rich insulating membrane. Myelination is a multi-step process initiated by axon-glia recognition triggering glial polarisation followed by targeted myelin membrane expansion and compaction. Thereby, a myelin sheath of complex subdomain structure is established. Continuous communication between neurons and glial cells is essential for myelin maintenance and axonal integrity. A diverse group of diseases, from multiple sclerosis to schizophrenia, have been linked to malfunction of myelinating cells reflecting the physiological importance of the axon-glial unit. This review describes the mechanisms of axonal signal integration by oligodendrocytes emphasising the central role of the Src-family kinase Fyn during CNS myelination. Furthermore, we discuss myelin membrane trafficking with particular focus on endocytic recycling and the control of PLP (proteolipid protein transport by SNARE proteins. Finally, PLP mistrafficking is considered in the context of myelin diseases.

  1. Differential compartmentalization of mRNAs in squid giant axon. (United States)

    Chun, J T; Gioio, A E; Crispino, M; Giuditta, A; Kaplan, B B


    Previously, we reported that the squid giant axon contains a heterogeneous population of mRNAs that includes beta-actin, beta-tubulin, kinesin, neurofilament proteins, and enolase. To define the absolute levels and relative distribution of these mRNAs, we have used competitive reverse transcription-PCR to quantify the levels of five mRNAs present in the giant axon and giant fiber lobe (GFL), the location of the parental cell soma. In the GFL, the number of transcripts for these mRNAs varied over a fourfold range, with beta-tubulin being the most abundant mRNA species (1.25 x 10(9) molecules per GFL). Based on transcript number, the rank order of mRNA levels in the GFL was beta-tubulin > beta-actin > kinesin > enolase > microtubule-associated protein (MAP) H1. In contrast, kinesin mRNA was most abundant in the axon (4.1 x 10(7) molecules per axon) with individual mRNA levels varying 15-fold. The rank order of mRNA levels in the axon was kinesin > beta-tubulin > MAP H1 > beta-actin > enolase. The relative abundance of the mRNA species in the axon did not correlate with the size of the transcript, nor was it directly related to their corresponding levels in the GFL. Taken together, these findings confirm that significant amounts of mRNA are present in the giant axon and suggest that specific mRNAs are differentially transported into the axonal domain.

  2. Intra-axonal protein synthesis - a new target for neural repair?

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    Jeffery L Twiss


    Full Text Available Although initially argued to be a feature of immature neurons with incomplete polarization, there is clear evidence that neurons in the peripheral nervous system retain the capacity for intra-axonal protein synthesis well into adulthood. This localized protein synthesis has been shown to contribute to injury signaling and axon regeneration in peripheral nerves. Recent works point to potential for protein synthesis in axons of the vertebrate central nervous system. mRNAs and protein synthesis machinery have now been documented in lamprey, mouse, and rat spinal cord axons. Intra-axonal protein synthesis appears to be activated in adult vertebrate spinal cord axons when they are regeneration-competent. Rat spinal cord axons regenerating into a peripheral nerve graft contain mRNAs and markers of activated translational machinery. Indeed, levels of some growth-associated mRNAs in these spinal cord axons are comparable to the regenerating sciatic nerve. Markers of active translation tend to decrease when these axons stop growing, but can be reactivated by a second axotomy. These emerging observations raise the possibility that mRNA transport into and translation within axons could be targeted to facilitate regeneration in both the peripheral and central nervous systems.

  3. Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon. (United States)

    Ma, Marek


    Axonal injury and degeneration, whether primary or secondary, contribute to the morbidity and mortality seen in many acquired and inherited central nervous system (CNS) and peripheral nervous system (PNS) disorders, such as traumatic brain injury, spinal cord injury, cerebral ischemia, neurodegenerative diseases, and peripheral neuropathies. The calpain family of proteases has been mechanistically linked to the dysfunction and degeneration of axons. While the direct mechanisms by which transection, mechanical strain, ischemia, or complement activation trigger intra-axonal calpain activity are likely different, the downstream effects of unregulated calpain activity may be similar in seemingly disparate diseases. In this review, a brief examination of axonal structure is followed by a focused overview of the calpain family. Finally, the mechanisms by which calpains may disrupt the axonal cytoskeleton, transport, and specialized domains (axon initial segment, nodes, and terminals) are discussed. © 2013.

  4. Reversible Axonal Dystrophy by Calcium Modulation in Frataxin-Deficient Sensory Neurons of YG8R Mice

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    Belén Mollá


    Full Text Available Friedreich’s ataxia (FRDA is a peripheral neuropathy involving a loss of proprioceptive sensory neurons. Studies of biopsies from patients suggest that axonal dysfunction precedes the death of proprioceptive neurons in a dying-back process. We observed that the deficiency of frataxin in sensory neurons of dorsal root ganglia (DRG of the YG8R mouse model causes the formation of axonal spheroids which retain dysfunctional mitochondria, shows alterations in the cytoskeleton and it produces impairment of axonal transport and autophagic flux. The homogenous distribution of axonal spheroids along the neurites supports the existence of continues focal damages. This lead us to propose for FRDA a model of distal axonopathy based on axonal focal damages. In addition, we observed the involvement of oxidative stress and dyshomeostasis of calcium in axonal spheroid formation generating axonal injury as a primary cause of pathophysiology. Axonal spheroids may be a consequence of calcium imbalance, thus we propose the quenching or removal extracellular Ca2+ to prevent spheroids formation. In our neuronal model, treatments with BAPTA and o-phenanthroline reverted the axonal dystrophy and the mitochondrial dysmorphic parameters. These results support the hypothesis that axonal pathology is reversible in FRDA by pharmacological manipulation of intracellular Ca2+ with Ca2+ chelators or metalloprotease inhibitors, preventing Ca2+-mediated axonal injury. Thus, the modulation of Ca2+ levels may be a relevant therapeutic target to develop early axonal protection and prevent dying-back neurodegeneration.

  5. Antidiabetic and Antihyperlipidemic Effects of Clitocybe nuda on Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

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    Mei-Hsing Chen


    Full Text Available The objective of this study was to evaluate the antihyperlipidemic and antihyperglycemic effects and mechanism of the extract of Clitocybe nuda (CNE, in high-fat- (HF- fed mice. C57BL/6J was randomly divided into two groups: the control (CON group was fed with a low-fat diet, whereas the experimental group was fed with a HF diet for 8 weeks. Then, the HF group was subdivided into five groups and was given orally CNE (including C1: 0.2, C2: 0.5, and C3: 1.0 g/kg/day extracts or rosiglitazone (Rosi or vehicle for 4 weeks. CNE effectively prevented HF-diet-induced increases in the levels of blood glucose, triglyceride, insulin (P<0.001, P<0.01, P<0.05, resp. and attenuated insulin resistance. By treatment with CNE, body weight gain, weights of white adipose tissue (WAT and hepatic triacylglycerol content were reduced; moreover, adipocytes in the visceral depots showed a reduction in size. By treatment with CNE, the protein contents of glucose transporter 4 (GLUT4 were significantly increased in C3-treated group in the skeletal muscle. Furthermore, CNE reduces the hepatic expression of glucose-6-phosphatase (G6Pase and glucose production. CNE significantly increases protein contents of phospho-AMP-activated protein kinase (AMPK in the skeletal muscle and adipose and liver tissues. Therefore, it is possible that the activation of AMPK by CNE leads to diminished gluconeogenesis in the liver and enhanced glucose uptake in skeletal muscle. It is shown that CNE exhibits hypolipidemic effect in HF-fed mice by increasing ATGL expression, which is known to help triglyceride to hydrolyze. Moreover, antidiabetic properties of CNE occurred as a result of decreased hepatic glucose production via G6Pase downregulation and improved insulin sensitization. Thus, amelioration of diabetic and dyslipidemic states by CNE in HF-fed mice occurred by regulation of GLUT4, G6Pase, ATGL, and AMPK phosphorylation.

  6. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila.

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    Jason E Duncan

    Full Text Available Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila. The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila, which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila, we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport.

  7. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila (United States)

    Duncan, Jason E.; Lytle, Nikki K.; Zuniga, Alfredo; Goldstein, Lawrence S. B.


    Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila . The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila , which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila , we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport. PMID:23840848

  8. Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus. (United States)

    Huang, Jialing; Lazear, Helen M; Friedman, Harvey M


    The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

    International Nuclear Information System (INIS)

    Huang Jialing; Lazear, Helen M.; Friedman, Harvey M.


    The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.

  10. Relationship of acute axonal damage, Wallerian degeneration, and clinical disability in multiple sclerosis. (United States)

    Singh, Shailender; Dallenga, Tobias; Winkler, Anne; Roemer, Shanu; Maruschak, Brigitte; Siebert, Heike; Brück, Wolfgang; Stadelmann, Christine


    Axonal damage and loss substantially contribute to the incremental accumulation of clinical disability in progressive multiple sclerosis. Here, we assessed the amount of Wallerian degeneration in brain tissue of multiple sclerosis patients in relation to demyelinating lesion activity and asked whether a transient blockade of Wallerian degeneration decreases axonal loss and clinical disability in a mouse model of inflammatory demyelination. Wallerian degeneration and acute axonal damage were determined immunohistochemically in the periplaque white matter of multiple sclerosis patients with early actively demyelinating lesions, chronic active lesions, and inactive lesions. Furthermore, we studied the effects of Wallerian degeneration blockage on clinical severity, inflammatory pathology, acute axonal damage, and long-term axonal loss in experimental autoimmune encephalomyelitis using Wallerian degeneration slow (Wld S ) mutant mice. The highest numbers of axons undergoing Wallerian degeneration were found in the perilesional white matter of multiple sclerosis patients early in the disease course and with actively demyelinating lesions. Furthermore, Wallerian degeneration was more abundant in patients harboring chronic active as compared to chronic inactive lesions. No co-localization of neuropeptide Y-Y1 receptor, a bona fide immunohistochemical marker of Wallerian degeneration, with amyloid precursor protein, frequently used as an indicator of acute axonal transport disturbance, was observed in human and mouse tissue, indicating distinct axon-degenerative processes. Experimentally, a delay of Wallerian degeneration, as observed in Wld S mice, did not result in a reduction of clinical disability or acute axonal damage in experimental autoimmune encephalomyelitis, further supporting that acute axonal damage as reflected by axonal transport disturbances does not share common molecular mechanisms with Wallerian degeneration. Furthermore, delaying Wallerian degeneration

  11. Modulation of repulsive forces between neurofilaments by sidearm phosphorylation

    International Nuclear Information System (INIS)

    Kumar, Sanjay; Hoh, Jan H.


    Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured 'sidearm' domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate-phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces

  12. CSPGs inhibit axon branching by impairing mitochondria-dependent regulation of actin dynamics and axonal translation. (United States)

    Sainath, Rajiv; Ketschek, Andrea; Grandi, Leah; Gallo, Gianluca


    Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin-dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria-dependent axonal translation of cortactin, an actin associated protein involved in branching. Finally, the inhibitory effects of CSPGs on axon branching, actin cytoskeletal dynamics and the axonal translation of cortactin are reversed by culturing neurons with acetyl-l-carnitine, which promotes mitochondrial respiration. Collectively these data indicate that CSPGs impair mitochondrial function in axons, an effect which contributes to the inhibition of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017. © 2016 Wiley Periodicals, Inc.

  13. Slowing of axonal regeneration is correlated with increased axonal viscosity during aging

    Directory of Open Access Journals (Sweden)

    Heidemann Steven R


    Full Text Available Abstract Background As we age, the speed of axonal regeneration declines. At the biophysical level, why this occurs is not well understood. Results To investigate we first measured the rate of axonal elongation of sensory neurons cultured from neonatal and adult rats. We found that neonatal axons grew 40% faster than adult axons (11.5 µm/hour vs. 8.2 µm/hour. To determine how the mechanical properties of axons change during maturation, we used force calibrated towing needles to measure the viscosity (stiffness and strength of substrate adhesion of neonatal and adult sensory axons. We found no significant difference in the strength of adhesions, but did find that adult axons were 3 times intrinsically stiffer than neonatal axons. Conclusions Taken together, our results suggest decreasing axonal stiffness may be part of an effective strategy to accelerate the regeneration of axons in the adult peripheral nervous system.

  14. Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation. (United States)

    di Penta, Alessandra; Moreno, Beatriz; Reix, Stephanie; Fernandez-Diez, Begoña; Villanueva, Maite; Errea, Oihana; Escala, Nagore; Vandenbroeck, Koen; Comella, Joan X; Villoslada, Pablo


    Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines

  15. The Kinesin Adaptor Calsyntenin-1 Organizes Microtubule Polarity and Regulates Dynamics during Sensory Axon Arbor Development

    Directory of Open Access Journals (Sweden)

    Mary C. Halloran


    Full Text Available Axon growth and branching, and development of neuronal polarity are critically dependent on proper organization and dynamics of the microtubule (MT cytoskeleton. MTs must organize with correct polarity for delivery of diverse cargos to appropriate subcellular locations, yet the molecular mechanisms regulating MT polarity remain poorly understood. Moreover, how an actively branching axon reorganizes MTs to direct their plus ends distally at branch points is unknown. We used high-speed, in vivo imaging of polymerizing MT plus ends to characterize MT dynamics in developing sensory axon arbors in zebrafish embryos. We find that axonal MTs are highly dynamic throughout development, and that the peripheral and central axons of sensory neurons show differences in MT behaviors. Furthermore, we show that Calsyntenin-1 (Clstn-1, a kinesin adaptor required for sensory axon branching, also regulates MT polarity in developing axon arbors. In wild type neurons the vast majority of MTs are directed in the correct plus-end-distal orientation from early stages of development. Loss of Clstn-1 causes an increase in MTs polymerizing in the retrograde direction. These misoriented MTs most often are found near growth cones and branch points, suggesting Clstn-1 is particularly important for organizing MT polarity at these locations. Together, our results suggest that Clstn-1, in addition to regulating kinesin-mediated cargo transport, also organizes the underlying MT highway during axon arbor development.

  16. Myosin-V Induces Cargo Immobilization and Clustering at the Axon Initial Segment

    Directory of Open Access Journals (Sweden)

    Anne F. J. Janssen


    Full Text Available The selective transport of different cargoes into axons and dendrites underlies the polarized organization of the neuron. Although it has become clear that the combined activity of different motors determines the destination and selectivity of transport, little is known about the mechanistic details of motor cooperation. For example, the exact role of myosin-V in opposing microtubule-based axon entries has remained unclear. Here we use two orthogonal chemically-induced heterodimerization systems to independently recruit different motors to cargoes. We find that recruiting myosin-V to kinesin-propelled cargoes at approximately equal numbers is sufficient to stall motility. Kinesin-driven cargoes entering the axon were arrested in the axon initial segment (AIS upon myosin-V recruitment and accumulated in distinct actin-rich hotspots. Importantly, unlike proposed previously, myosin-V did not return these cargoes to the cell body, suggesting that additional mechanism are required to establish cargo retrieval from the AIS.

  17. Progress of Research on Diffuse Axonal Injury after Traumatic Brain Injury

    Directory of Open Access Journals (Sweden)

    Junwei Ma


    Full Text Available The current work reviews the concept, pathological mechanism, and process of diagnosing of DAI. The pathological mechanism underlying DAI is complicated, including axonal breakage caused by axonal retraction balls, discontinued protein transport along the axonal axis, calcium influx, and calpain-mediated hydrolysis of structural protein, degradation of axonal cytoskeleton network, the changes of transport proteins such as amyloid precursor protein, and changes of glia cells. Based on the above pathological mechanism, the diagnosis of DAI is usually made using methods such as CT, traditional and new MRI, biochemical markers, and neuropsychological assessment. This review provides a basis in literature for further investigation and discusses the pathological mechanism. It may also facilitate improvement of the accuracy of diagnosis for DAI, which may come to play a critical role in breaking through the bottleneck of the clinical treatment of DAI and improving the survival and quality of life of patients through clear understanding of pathological mechanisms and accurate diagnosis.

  18. Cross-talk between KLF4 and STAT3 regulates axon regeneration (United States)

    Qin, Song; Zou, Yuhua; Zhang, Chun-Li


    Cytokine-induced activation of signal transducer and activator of transcription 3 (STAT3) promotes the regrowth of damaged axons in the adult central nervous system (CNS). Here we show that KLF4 physically interacts with STAT3 upon cytokine-induced phosphorylation of tyrosine 705 (Y705) on STAT3. This interaction suppresses STAT3-dependent gene expression by blocking its DNA-binding activity. The deletion of KLF4 in vivo induces axon regeneration of adult retinal ganglion cells (RGCs) via Janus kinase (JAK)-STAT3 signalling. This regeneration can be greatly enhanced by exogenous cytokine treatment, or removal of an endogenous JAK-STAT3 pathway inhibitor called suppressor of cytokine signalling 3 (SOCS3). These findings reveal an unexpected cross-talk between KLF4 and activated STAT3 in the regulation of axon regeneration that might have therapeutic implications in promoting repair of injured adult CNS.

  19. Quantifying mechanical force in axonal growth and guidance

    Directory of Open Access Journals (Sweden)

    Ahmad Ibrahim Mahmoud Athamneh


    Full Text Available Mechanical force plays a fundamental role in neuronal development, physiology, and regeneration. In particular, research has shown that force is involved in growth cone-mediated axonal growth and guidance as well as stretch-induced elongation when an organism increases in size after forming initial synaptic connections. However, much of the details about the exact role of force in these fundamental processes remain unknown. In this review, we highlight (1 standing questions concerning the role of mechanical force in axonal growth and guidance and (2 different experimental techniques used to quantify forces in axons and growth cones. We believe that satisfying answers to these questions will require quantitative information about the relationship between elongation, forces, cytoskeletal dynamics, axonal transport, signaling, substrate adhesion, and stiffness contributing to directional growth advance. Furthermore, we address why a wide range of force values have been reported in the literature, and what these values mean in the context of neuronal mechanics. We hope that this review will provide a guide for those interested in studying the role of force in development and regeneration of neuronal networks.

  20. Numerical analysis of the method of internal dialysis of giant axons


    Horn, L.W.


    This paper presents a numerical analysis of the method of internal dialysis used for studies of membrane transport in giant axons. Account is taken of the complete geometry, end effects, and finite dialyzate flow rates. Both influx and efflux experimental conditions are considered. Results place quantitative limits on system performance that are sufficiently general for use in experimental design. The completeness of solute equilibration and the uniformity of solute concentration at the axon ...

  1. Analytical investigation of various regimes of retrograde trafficking of neurotropic viruses in axons (United States)

    Kuznetsov, Andrey V.


    A model of retrograde axonal transport of neurotropic viruses is developed. The model accounts for active viral transport by dynein motors as well as for passive transport by diffusion; the destruction of the virus as it propagates toward the neuron soma is modeled utilizing a first-order decay rate process. The effect of a limited time during which the axonal synapse is exposed to the virus is incorporated. An analytical solution is obtained. The obtained solution makes it possible to identify four different regimes of viral transport in the axon that correspond to the following situations: (1) Small viral diffusivity and small rate of viral destruction; (2) Large viral diffusivity and small rate of viral destruction; (3) Small viral diffusivity and large rate of viral destruction; (4) Large viral diffusivity and large rate of viral destruction. Characteristic features of these regimes are discussed.

  2. Quantitative analysis of microtubule transport in growing nerve processes

    DEFF Research Database (Denmark)

    Ma*, Ytao; Shakiryanova*, Dinara; Vardya, Irina


    the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required...

  3. Parkinson's disease associated with impaired oxidative phosphorylation

    International Nuclear Information System (INIS)

    Finsterer, J.; Jarius, C.; Baumgartner, M.


    Parkinson's disease may be due to primary or secondary oxidative phosphorylation (OXPHOS) defects. In a 76-year-old man with Parkinson's disease since 1992, slightly but recurrently elevated creatine phosphokinase, recurrently elevated blood glucose, thickening of the left ventricular myocardium, bifascicular block and hypacusis were found. Cerebral MRI showed atrophy, periventricular demyelination, multiple, disseminated, supra- and infratentorial lacunas, and haemosiderin deposits in both posterior horns. Muscle biopsy showed typical features of an OXPHOS defect. Whether the association of Parkinson's disease and impaired OXPHOS was causative or coincidental remains unknown. Possibly, the mitochondrial defect acted as an additional risk factor for Parkinson's disease or the OXPHOS defect worsened the preexisting neurological impairments by a cumulative or synergistic mechanism. In conclusion, this case shows that Parkinson's disease may be associated with a mitochondrially or nuclearly encoded OXPHOS defect, manifesting as hypacusis, myopathy, axonal polyneuropathy, cardiomyopathy and recurrent subclinical ischaemic strokes and haemorrhages. (orig.)

  4. Formation of compact myelin is required for maturation of the axonal cytoskeleton (United States)

    Brady, S. T.; Witt, A. S.; Kirkpatrick, L. L.; de Waegh, S. M.; Readhead, C.; Tu, P. H.; Lee, V. M.


    Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons.

  5. Radial glia phagocytose axonal debris from degenerating overextending axons in the developing olfactory bulb. (United States)

    Amaya, Daniel A; Wegner, Michael; Stolt, C Claus; Chehrehasa, Fatemeh; Ekberg, Jenny A K; St John, James A


    Axon targeting during the development of the olfactory system is not always accurate, and numerous axons overextend past the target layer into the deeper layers of the olfactory bulb. To date, the fate of the mis-targeted axons has not been determined. We hypothesized that following overextension, the axons degenerate, and cells within the deeper layers of the olfactory bulb phagocytose the axonal debris. We utilized a line of transgenic mice that expresses ZsGreen fluorescent protein in primary olfactory axons. We found that overextending axons closely followed the filaments of radial glia present in the olfactory bulb during embryonic development. Following overextension into deeper layers of the olfactory bulb, axons degenerated and radial glia responded by phagocytosing the resulting debris. We used in vitro analysis to confirm that the radial glia had phagocytosed debris from olfactory axons. We also investigated whether the fate of overextending axons was altered when the development of the olfactory bulb was perturbed. In mice that lacked Sox10, a transcription factor essential for normal olfactory bulb development, we observed a disruption to the morphology and positioning of radial glia and an accumulation of olfactory axon debris within the bulb. Our results demonstrate that during early development of the olfactory system, radial glia play an important role in removing overextended axons from the deeper layers of the olfactory bulb. © 2014 Wiley Periodicals, Inc.

  6. Action Potential Dynamics in Fine Axons Probed with an Axonally Targeted Optical Voltage Sensor. (United States)

    Ma, Yihe; Bayguinov, Peter O; Jackson, Meyer B


    The complex and malleable conduction properties of axons determine how action potentials propagate through extensive axonal arbors to reach synaptic terminals. The excitability of axonal membranes plays a major role in neural circuit function, but because most axons are too thin for conventional electrical recording, their properties remain largely unexplored. To overcome this obstacle, we used a genetically encoded hybrid voltage sensor (hVOS) harboring an axonal targeting motif. Expressing this probe in transgenic mice enabled us to monitor voltage changes optically in two populations of axons in hippocampal slices, the large axons of dentate granule cells (mossy fibers) in the stratum lucidum of the CA3 region and the much finer axons of hilar mossy cells in the inner molecular layer of the dentate gyrus. Action potentials propagated with distinct velocities in each type of axon. Repetitive firing broadened action potentials in both populations, but at an intermediate frequency the degree of broadening differed. Repetitive firing also attenuated action potential amplitudes in both mossy cell and granule cell axons. These results indicate that the features of use-dependent action potential broadening, and possible failure, observed previously in large nerve terminals also appear in much finer unmyelinated axons. Subtle differences in the frequency dependences could influence the propagation of activity through different pathways to excite different populations of neurons. The axonally targeted hVOS probe used here opens up the diverse repertoire of neuronal processes to detailed biophysical study.

  7. AxonSeg: Open Source Software for Axon and Myelin Segmentation and Morphometric Analysis. (United States)

    Zaimi, Aldo; Duval, Tanguy; Gasecka, Alicja; Côté, Daniel; Stikov, Nikola; Cohen-Adad, Julien


    Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy (SEM) only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLAB-based graphical user interface (GUI) and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing; (ii) pre-segmentation of axons over a cropped image and discriminant analysis (DA) to select the best parameters based on axon shape and intensity information; (iii) automatic axon and myelin segmentation over the full image; and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), SEM and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology. AxonSeg is open source and freely available at:

  8. Inner membrane fusion mediates spatial distribution of axonal mitochondria (United States)

    Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge


    In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution. PMID:26742817

  9. Regulating Axonal Responses to Injury: The Intersection between Signaling Pathways Involved in Axon Myelination and The Inhibition of Axon Regeneration (United States)

    Rao, Sudheendra N. R.; Pearse, Damien D.


    Following spinal cord injury (SCI), a multitude of intrinsic and extrinsic factors adversely affect the gene programs that govern the expression of regeneration-associated genes (RAGs) and the production of a diversity of extracellular matrix molecules (ECM). Insufficient RAG expression in the injured neuron and the presence of inhibitory ECM at the lesion, leads to structural alterations in the axon that perturb the growth machinery, or form an extraneous barrier to axonal regeneration, respectively. Here, the role of myelin, both intact and debris, in antagonizing axon regeneration has been the focus of numerous investigations. These studies have employed antagonizing antibodies and knockout animals to examine how the growth cone of the re-growing axon responds to the presence of myelin and myelin-associated inhibitors (MAIs) within the lesion environment and caudal spinal cord. However, less attention has been placed on how the myelination of the axon after SCI, whether by endogenous glia or exogenously implanted glia, may alter axon regeneration. Here, we examine the intersection between intracellular signaling pathways in neurons and glia that are involved in axon myelination and axon growth, to provide greater insight into how interrogating this complex network of molecular interactions may lead to new therapeutics targeting SCI. PMID:27375427

  10. IMP2 axonal localization, RNA interactome, and function in the development of axon trajectories

    DEFF Research Database (Denmark)

    Preitner, Nicolas; Quan, Jie; Li, Xinmin


    RNA-based regulatory mechanisms play important roles in the development and plasticity of neural circuits and neurological disease. Developing axons provide a model well suited to the study of RNA-based regulation, and contain specific subsets of mRNAsthat are locally translated and have roles...... to strong defects in commissural axon trajectories at the midline intermediate target. These results reveal a highly distinctive axonal enrichment of IMP2, show that it interacts with a network of axon guidance-related mRNAs, and reveal that it is required for normal axon pathfinding during vertebrate...

  11. Squid Giant Axons Synthesize NF Proteins. (United States)

    Crispino, Marianna; Chun, Jong Tai; Giuditta, Antonio


    Squid giant axon has been an excellent model system for studying fundamental topics in neurobiology such as neuronal signaling. It has been also useful in addressing the questions of local protein synthesis in the axons. Incubation of isolated squid giant axons with [ 35 S]methionine followed by immunoprecipitation with a rabbit antibody against all squid neurofilament (NF) proteins demonstrates the local synthesis of a major 180 kDa NF protein and of several NF proteins of lower molecular weights. Their identification as NF proteins is based on their absence in the preimmune precipitates. Immunoprecipitates washed with more stringent buffers confirmed these results. Our data are at variance with a recent study based on the same experimental procedure that failed to visualize the local synthesis of NF proteins by the giant axon and thereby suggested their exclusive derivation from nerve cell bodies (as reported by Gainer et al. in Cell Mol Neurobiol 37:475-486, 2017). By reviewing the pertinent literature, we confute the claims that mRNA translation is absent in mature axons because of a putative translation block and that most proteins of mature axons are synthesized in the surrounding glial cells. Given the intrinsic axonal capacity to synthesize proteins, we stress the glial derivation of axonal and presynaptic RNAs and the related proposal that these neuronal domains are endowed with largely independent gene expression systems (as reported by Giuditta et al. in Physiol Rev 88:515-555, 2008).

  12. Ascending Midbrain Dopaminergic Axons Require Descending GAD65 Axon Fascicles for Normal Pathfinding

    Directory of Open Access Journals (Sweden)

    Claudia Marcela Garcia-Peña


    Full Text Available The Nigrostriatal pathway (NSP is formed by dopaminergic axons that project from the ventral midbrain to the dorsolateral striatum as part of the medial forebrain bundle. Previous studies have implicated chemotropic proteins in the formation of the NSP during development but little is known of the role of substrate-anchored signals in this process. We observed in mouse and rat embryos that midbrain dopaminergic axons ascend in close apposition to descending GAD65-positive axon bundles throughout their trajectory to the striatum. To test whether such interaction is important for dopaminergic axon pathfinding, we analyzed transgenic mouse embryos in which the GAD65 axon bundle was reduced by the conditional expression of the diphtheria toxin. In these embryos we observed dopaminergic misprojection into the hypothalamic region and abnormal projection in the striatum. In addition, analysis of Robo1/2 and Slit1/2 knockout embryos revealed that the previously described dopaminergic misprojection in these embryos is accompanied by severe alterations in the GAD65 axon scaffold. Additional studies with cultured dopaminergic neurons and whole embryos suggest that NCAM and Robo proteins are involved in the interaction of GAD65 and dopaminergic axons. These results indicate that the fasciculation between descending GAD65 axon bundles and ascending dopaminergic axons is required for the stereotypical NSP formation during brain development and that known guidance cues may determine this projection indirectly by instructing the pathfinding of the axons that are part of the GAD65 axon scaffold.

  13. The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans (United States)

    Chisholm, Andrew D.; Hutter, Harald; Jin, Yishi; Wadsworth, William G.


    The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment. PMID:28114100

  14. Meninges-derived cues control axon guidance. (United States)

    Suter, Tracey A C S; DeLoughery, Zachary J; Jaworski, Alexander


    The axons of developing neurons travel long distances along stereotyped pathways under the direction of extracellular cues sensed by the axonal growth cone. Guidance cues are either secreted proteins that diffuse freely or bind the extracellular matrix, or membrane-anchored proteins. Different populations of axons express distinct sets of receptors for guidance cues, which results in differential responses to specific ligands. The full repertoire of axon guidance cues and receptors and the identity of the tissues producing these cues remain to be elucidated. The meninges are connective tissue layers enveloping the vertebrate brain and spinal cord that serve to protect the central nervous system (CNS). The meninges also instruct nervous system development by regulating the generation and migration of neural progenitors, but it has not been determined whether they help guide axons to their targets. Here, we investigate a possible role for the meninges in neuronal wiring. Using mouse neural tissue explants, we show that developing spinal cord meninges produce secreted attractive and repulsive cues that can guide multiple types of axons in vitro. We find that motor and sensory neurons, which project axons across the CNS-peripheral nervous system (PNS) boundary, are attracted by meninges. Conversely, axons of both ipsi- and contralaterally projecting dorsal spinal cord interneurons are repelled by meninges. The responses of these axonal populations to the meninges are consistent with their trajectories relative to meninges in vivo, suggesting that meningeal guidance factors contribute to nervous system wiring and control which axons are able to traverse the CNS-PNS boundary. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. A macroscopic model of traffic jams in axons. (United States)

    Kuznetsov, A V; Avramenko, A A


    The purpose of this paper is to develop a minimal macroscopic model capable of explaining the formation of traffic jams in fast axonal transport. The model accounts for the decrease of the number density of positively (and negatively) oriented microtubules near the location of the traffic jam due to formation of microtubule swirls; the model also accounts for the reduction of the effective velocity of organelle transport in the traffic jam region due to organelles falling off microtubule tracks more often in the swirl region. The model is based on molecular-motor-assisted transport equations and the hydrodynamic model of traffic jams in highway traffic. Parametric analyses of the model's predictions for various values of viscosity of the traffic flow, variance of the velocity distribution, diffusivity of microtubule-bound and free organelles, rate constants for binding to and detachment from microtubules, relaxation time, and average motor velocities of the retrograde and anterograde transport, are carried out.

  16. Developmental downregulation of LIS1 expression limits axonal extension and allows axon pruning

    Directory of Open Access Journals (Sweden)

    Kanako Kumamoto


    Full Text Available The robust axonal growth and regenerative capacities of young neurons decrease substantially with age. This developmental downregulation of axonal growth may facilitate axonal pruning and neural circuit formation but limits functional recovery following nerve damage. While external factors influencing axonal growth have been extensively investigated, relatively little is known about the intrinsic molecular changes underlying the age-dependent reduction in regeneration capacity. We report that developmental downregulation of LIS1 is responsible for the decreased axonal extension capacity of mature dorsal root ganglion (DRG neurons. In contrast, exogenous LIS1 expression or endogenous LIS1 augmentation by calpain inhibition restored axonal extension capacity in mature DRG neurons and facilitated regeneration of the damaged sciatic nerve. The insulator protein CTCF suppressed LIS1 expression in mature DRG neurons, and this reduction resulted in excessive accumulation of phosphoactivated GSK-3β at the axon tip, causing failure of the axonal extension. Conversely, sustained LIS1 expression inhibited developmental axon pruning in the mammillary body. Thus, LIS1 regulation may coordinate the balance between axonal growth and pruning during maturation of neuronal circuits.

  17. Dynamics of target recognition by interstitial axon branching along developing cortical axons. (United States)

    Bastmeyer, M; O'Leary, D D


    Corticospinal axons innervate their midbrain, hindbrain, and spinal targets by extending collateral branches interstitially along their length. To establish that the axon shaft rather than the axonal growth cone is responsible for target recognition in this system, and to characterize the dynamics of interstitial branch formation, we have studied this process in an in vivo-like setting using slice cultures from neonatal mice containing the entire pathway of corticospinal axons. Corticospinal axons labeled with the dye 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (or Dil) were imaged using time-lapse video microscopy of their pathway overlying the basilar pons, their major hindbrain target. The axon shaft millimeters behind the growth cone exhibits several dynamic behaviors, including the de novo formation of varicosities and filopodia-like extensions, and a behavior that we term "pulsation," which is characterized by a variable thickening and thining of short segments of the axon. An individual axon can have multiple sites of branching activity, with many of the branches being transient. These dynamic behaviors occur along the portion of the axon shaft overlying the basilar pons, but not just caudal to it. Once the collaterals extend into the pontine neuropil, they branch further in the neuropil, while the parent axon becomes quiescent. Thus, the branching activity is spatially restricted to specific portions of the axon, as well as temporally restricted to a relatively brief time window. These findings provide definitive evidence that collateral branches form de novo along corticospinal axons and establish that the process of target recognition in this system is a property of the axon shaft rather than the leading growth cone.

  18. Local gene expression in axons and nerve endings: the glia-neuron unit. (United States)

    Giuditta, Antonio; Chun, Jong Tai; Eyman, Maria; Cefaliello, Carolina; Bruno, Anna Paola; Crispino, Marianna


    Neurons have complex and often extensively elongated processes. This unique cell morphology raises the problem of how remote neuronal territories are replenished with proteins. For a long time, axonal and presynaptic proteins were thought to be exclusively synthesized in the cell body, which delivered them to peripheral sites by axoplasmic transport. Despite this early belief, protein has been shown to be synthesized in axons and nerve terminals, substantially alleviating the trophic burden of the perikaryon. This observation raised the question of the cellular origin of the peripheral RNAs involved in protein synthesis. The synthesis of these RNAs was initially attributed to the neuron soma almost by default. However, experimental data and theoretical considerations support the alternative view that axonal and presynaptic RNAs are also transcribed in the flanking glial cells and transferred to the axon domain of mature neurons. Altogether, these data suggest that axons and nerve terminals are served by a distinct gene expression system largely independent of the neuron cell body. Such a local system would allow the neuron periphery to respond promptly to environmental stimuli. This view has the theoretical merit of extending to axons and nerve terminals the marginalized concept of a glial supply of RNA (and protein) to the neuron cell body. Most long-term plastic changes requiring de novo gene expression occur in these domains, notably in presynaptic endings, despite their intrinsic lack of transcriptional capacity. This review enlightens novel perspectives on the biology and pathobiology of the neuron by critically reviewing these issues.

  19. Mouse Intermittent Hypoxia Mimicking Apnea of Prematurity: Effects on Myelinogenesis and Axonal Maturation (United States)



    Premature babies are at high risk for both infantile apnea and long-term neurobehavioral deficits. Recent studies suggest that diffuse structural changes in brain white matter are a positive predictor of poor cognitive outcomes. Since oligodendrocyte maturation, myelination, axon development and synapse formation mainly occur in the 3rd trimester of gestation and 1st postnatal year, infantile apnea could lead to and/or exaggerate white matter impairments in preterm neonates. Therefore, we investigated oligodendroglia and axon development in a neonatal mouse model of intermittent hypoxia between postnatal days 2 to 10. During critical phases of central nervous system development, intermittent hypoxia induced hypomyelination in the corpus callosum, striatum, fornix and cerebellum, but not the pons or spinal cord. Intermittent hypoxia-elicited alterations in myelin-forming processes were reflected by decreased expression of myelin proteins, including MBP, PLP, MAG and CNPase, possibly due to arrested maturation of oligodendrocytes. Ultra-structural abnormalities were apparent in the myelin sheath and axon. Immature oligodendrocytes were more vulnerable to neonatal intermittent hypoxia exposures than developing axons, suggesting that hypomyelination may contribute, at least partially, to axonal deficits. Insufficient neurofilament synthesis with anomalous components of neurofilament subunits, β-tubulin and MAP2 isoforms indicated immaturity of axons in intermittent hypoxia-exposed mouse brains. In addition, down-regulation of Synapsin I, Synaptophysin and Gap-43 phosphorylation suggested a potential stunt in axonogenesis and synaptogenesis. The region-selective and complex impairment in brain white matter induced by intermittent hypoxia was further associated with electrophysiological changes that may underlie long-term neurobehavioral sequelae. PMID:21953180

  20. Genetics Home Reference: giant axonal neuropathy (United States)

    ... connect the brain and spinal cord (central nervous system) to muscles and to sensory cells that detect sensations such as touch, pain, heat, and sound. However, axons in the central nervous system are affected as well. The signs and symptoms ...

  1. Drug therapy for chronic idiopathic axonal polyneuropathy

    NARCIS (Netherlands)

    Vrancken, A. F. J. E.; van Schaik, I. N.; Hughes, R. A. C.; Notermans, N. C.


    BACKGROUND: Chronic idiopathic axonal polyneuropathy is an insidiously progressive sensory or sensorimotor polyneuropathy that affects elderly people. Although severe disability or handicap does not occur, it reduces quality of life. OBJECTIVES: To assess whether drug therapy for chronic idiopathic

  2. hnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons. (United States)

    Briese, Michael; Saal-Bauernschubert, Lena; Ji, Changhe; Moradi, Mehri; Ghanawi, Hanaa; Uhl, Michael; Appenzeller, Silke; Backofen, Rolf; Sendtner, Michael


    Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance. Copyright © 2018 the Author(s). Published by PNAS.

  3. Rosiglitazone ameliorates diffuse axonal injury by reducing loss of tau and up-regulating caveolin-1 expression

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    Yong-lin Zhao


    Full Text Available Rosiglitazone up-regulates caveolin-1 levels and has neuroprotective effects in both chronic and acute brain injury. Therefore, we postulated that rosiglitazone may ameliorate diffuse axonal injury via its ability to up-regulate caveolin-1, inhibit expression of amyloid-beta precursor protein, and reduce the loss and abnormal phosphorylation of tau. In the present study, intraperitoneal injection of rosiglitazone significantly reduced the levels of amyloid-beta precursor protein and hyperphosphorylated tau (phosphorylated at Ser 404 (p-tau (S 404 , and it increased the expression of total tau and caveolin-1 in the rat cortex. Our results show that rosiglitazone inhibits the expression of amyloid-beta precursor protein and lowers p-tau (S 404 levels, and it reduces the loss of total tau, possibly by up-regulating caveolin-1. These actions of rosiglitazone may underlie its neuroprotective effects in the treatment of diffuse axonal injury.

  4. Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy

    NARCIS (Netherlands)

    Kole, Maarten H. P.; Letzkus, Johannes J.; Stuart, Greg J.


    Action potentials are binary signals that transmit information via their rate and temporal pattern. In this context, the axon is thought of as a transmission line, devoid of a role in neuronal computation. Here, we show a highly localized role of axonal Kv1 potassium channels in shaping the action

  5. Axonal cleaved caspase-3 regulates axon targeting and morphogenesis in the developing auditory brainstem

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    Sarah E Rotschafer


    Full Text Available Caspase-3 is a cysteine protease that is most commonly associated with cell death. Recent studies have shown additional roles in mediating cell differentiation, cell proliferation, and development of cell morphology. We investigated the role of caspase-3 in the development of chick auditory brainstem nuclei during embryogenesis. Immunofluorescence from embryonic days E6-13 revealed that the temporal expression of cleaved caspase-3 follows the ascending anatomical pathway. Expression is first seen in the auditory portion of VIIIth nerve including central axonal regions projecting to nucleus magnocellularis (NM, then later in NM axons projecting to nucleus laminaris (NL, and subsequently in NL dendrites. To examine the function of cleaved caspase-3 in chick auditory brainstem development, we blocked caspase-3 cleavage in developing chick embryos with the caspase-3 inhibitor Z-DEVD-FMK from E6 to E9, then examined NM and NL morphology and NM axonal targeting on E10. NL lamination in treated embryos was disorganized and the neuropil around NL contained a significant number of glial cells normally excluded from this region. Additionally, NM axons projected into inappropriate portions of NL in Z-DEVD-FMK treated embyros. We found that the presence of misrouted axons was associated with more severe NL disorganization. The effects of axonal caspase-3 inhibition on both NL morphogenesis and NM axon targeting suggest that these developmental processes are coordinated, likely through communication between axons and their targets.

  6. (−-Epicatechin-3-O-β-d-allopyranoside from Davallia formosana, Prevents Diabetes and Hyperlipidemia by Regulation of Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

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    Chun-Ching Shih


    Full Text Available The purpose of this experiment was to determine the antidiabetic and lipid-lowering effects of (−-epicatechin-3-O-β-d-allopyranoside (BB from the roots and stems of Davallia formosana in mice. Animal treatment was induced by high-fat diet (HFD or low-fat diet (control diet, CD. After eight weeks of HFD or CD exposure, the HFD mice were treating with BB or rosiglitazone (Rosi or fenofibrate (Feno or water through gavage for another four weeks. However, at 12 weeks, the HFD-fed group had enhanced blood levels of glucose, triglyceride (TG, and insulin. BB treatment significantly decreased blood glucose, TG, and insulin levels. Moreover, visceral fat weights were enhanced in HFD-fed mice, accompanied by increased blood leptin concentrations and decreased adiponectin levels, which were reversed by treatment with BB. Muscular membrane protein levels of glucose transporter 4 (GLUT4 were reduced in HFD-fed mice and significantly enhanced upon administration of BB, Rosi, and Feno. Moreover, BB treatment markedly increased hepatic and skeletal muscular expression levels of phosphorylation of AMP-activated (adenosine monophosphate protein kinase (phospho-AMPK. BB also decreased hepatic mRNA levels of phosphenolpyruvate carboxykinase (PEPCK, which are associated with a decrease in hepatic glucose production. BB-exerted hypotriglyceridemic activity may be partly associated with increased mRNA levels of peroxisome proliferator activated receptor α (PPARα, and with reduced hepatic glycerol-3-phosphate acyltransferase (GPAT mRNA levels in the liver, which decreased triacylglycerol synthesis. Nevertheless, we demonstrated BB was a useful approach for the management of type 2 diabetes and dyslipidemia in this animal model.

  7. Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting (United States)

    Müller, Oliver; Ivanova, Lyudmila; Bialy, Dagmara; Pohlmann, Anja; Binz, Anne; Hegemann, Maike; Viejo-Borbolla, Abel; Rosenhahn, Bodo; Bauerfeind, Rudolf; Sodeik, Beate


    Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells. PMID:29284065

  8. Increased mitochondrial content in remyelinated axons: implications for multiple sclerosis (United States)

    Zambonin, Jessica L.; Zhao, Chao; Ohno, Nobuhiko; Campbell, Graham R.; Engeham, Sarah; Ziabreva, Iryna; Schwarz, Nadine; Lee, Sok Ee; Frischer, Josa M.; Turnbull, Doug M.; Trapp, Bruce D.; Lassmann, Hans; Franklin, Robin J. M.


    Mitochondrial content within axons increases following demyelination in the central nervous system, presumably as a response to the changes in energy needs of axons imposed by redistribution of sodium channels. Myelin sheaths can be restored in demyelinated axons and remyelination in some multiple sclerosis lesions is extensive, while in others it is incomplete or absent. The effects of remyelination on axonal mitochondrial content in multiple sclerosis, particularly whether remyelination completely reverses the mitochondrial changes that follow demyelination, are currently unknown. In this study, we analysed axonal mitochondria within demyelinated, remyelinated and myelinated axons in post-mortem tissue from patients with multiple sclerosis and controls, as well as in experimental models of demyelination and remyelination, in vivo and in vitro. Immunofluorescent labelling of mitochondria (porin, a voltage-dependent anion channel expressed on all mitochondria) and axons (neurofilament), and ultrastructural imaging showed that in both multiple sclerosis and experimental demyelination, mitochondrial content within remyelinated axons was significantly less than in acutely and chronically demyelinated axons but more numerous than in myelinated axons. The greater mitochondrial content within remyelinated, compared with myelinated, axons was due to an increase in density of porin elements whereas increase in size accounted for the change observed in demyelinated axons. The increase in mitochondrial content in remyelinated axons was associated with an increase in mitochondrial respiratory chain complex IV activity. In vitro studies showed a significant increase in the number of stationary mitochondria in remyelinated compared with myelinated and demyelinated axons. The number of mobile mitochondria in remyelinated axons did not significantly differ from myelinated axons, although significantly greater than in demyelinated axons. Our neuropathological data and findings in

  9. Molecular cloning and characterization of a novel mRNA present in the squid giant axon. (United States)

    Chun, J T; Gioio, A E; Crispino, M; Eyman, M; Giuditta, A; Kaplan, B B


    Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT-PCR analysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (approximately 0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative protein contains a single transmembrane domain located in the middle of the molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphorylation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channel protein.

  10. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    Directory of Open Access Journals (Sweden)

    Yueh-Hsiung Kuo


    Full Text Available This study investigated the potential effects of dehydroeburicoic acid (TT, a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom on membrane glucose transporter 4 (GLUT4 and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4 and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels, fenofibrate (Feno (at 0.25 g/kg body weight, metformin (Metf (at 0.3 g/kg body weight or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%. TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase, an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator

  11. Guidance of retinal axons in mammals. (United States)

    Herrera, Eloísa; Erskine, Lynda; Morenilla-Palao, Cruz


    In order to navigate through the surrounding environment many mammals, including humans, primarily rely on vision. The eye, composed of the choroid, sclera, retinal pigmented epithelium, cornea, lens, iris and retina, is the structure that receives the light and converts it into electrical impulses. The retina contains six major types of neurons involving in receiving and modifying visual information and passing it onto higher visual processing centres in the brain. Visual information is relayed to the brain via the axons of retinal ganglion cells (RGCs), a projection known as the optic pathway. The proper formation of this pathway during development is essential for normal vision in the adult individual. Along this pathway there are several points where visual axons face 'choices' in their direction of growth. Understanding how these choices are made has advanced significantly our knowledge of axon guidance mechanisms. Thus, the development of the visual pathway has served as an extremely useful model to reveal general principles of axon pathfinding throughout the nervous system. However, due to its particularities, some cellular and molecular mechanisms are specific for the visual circuit. Here we review both general and specific mechanisms involved in the guidance of mammalian RGC axons when they are traveling from the retina to the brain to establish precise and stereotyped connections that will sustain vision. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Phosphorylation site prediction in plants. (United States)

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong


    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community.

  13. Brimonidine prevents axonal and somatic degeneration of retinal ganglion cell neurons

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    Crish Samuel D


    Full Text Available Abstract Background Brimonidine is a common drug for lowering ocular pressure and may directly protect retinal ganglion cells in glaucoma. The disease involves early loss of retinal ganglion cell transport to brain targets followed by axonal and somatic degeneration. We examined whether brimonidine preserves ganglion cell axonal transport and abates degeneration in rats with elevated ocular pressure induced by laser cauterization of the episcleral veins. Results Ocular pressure was elevated unilaterally by 90% for a period of 8 weeks post- cauterization. During this time, brimonidine (1mg/kg/day or vehicle (phosphate-buffered saline was delivered systemically and continuously via subcutaneous pump. Animals received bilateral intravitreal injections of fluorescent cholera toxin subunit β (CTB two days before sacrifice to assess anterograde transport. In retinas from the vehicle group, elevated pressure induced a 44% decrease in the fraction of ganglion cells with intact uptake of CTB and a 14-42% reduction in the number of immuno-labelled ganglion cell bodies, with the worst loss occurring nasally. Elevated pressure also caused a 33% loss of ganglion cell axons in vehicle optic nerves and a 70% decrease in CTB transport to the superior colliculus. Each of these components of ganglion cell degeneration was either prevented or significantly reduced in the brimonidine treatment group. Conclusions Continuous and systemic treatment with brimonidine by subcutaneous injection significantly improved retinal ganglion cell survival with exposure to elevated ocular pressure. This effect was most striking in the nasal region of the retina. Brimonidine treatment also preserved ganglion cell axon morphology, sampling density and total number in the optic nerve with elevated pressure. Consistent with improved outcome in the optic projection, brimonidine also significantly reduced the deficits in axonal transport to the superior colliculus associated with

  14. Cholesterol Perturbation in Mice Results in p53 Degradation and Axonal Pathology through p38 MAPK and Mdm2 Activation.

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    Qingyu Qin

    Full Text Available Perturbation of lipid metabolism, especially of cholesterol homeostasis, can be catastrophic to mammalian brain, as it has the highest level of cholesterol in the body. This notion is best illustrated by the severe progressive neurodegeneration in Niemann-Pick Type C (NPC disease, one of the lysosomal storage diseases, caused by mutations in the NPC1 or NPC2 gene. In this study, we found that growth cone collapse induced by genetic or pharmacological disruption of cholesterol egress from late endosomes/lysosomes was directly related to a decrease in axonal and growth cone levels of the phosphorylated form of the tumor suppressor factor p53. Cholesterol perturbation-induced growth cone collapse and decrease in phosphorylated p53 were reduced by inhibition of p38 mitogen-activated protein kinase (MAPK and murine double minute (Mdm2 E3 ligase. Growth cone collapse induced by genetic (npc1-/- or pharmacological modification of cholesterol metabolism was Rho kinase (ROCK-dependent and associated with increased RhoA protein synthesis; both processes were significantly reduced by P38 MAPK or Mdm2 inhibition. Finally, in vivo ROCK inhibition significantly increased phosphorylated p53 levels and neurofilaments in axons, and axonal bundle size in npc1-/- mice. These results indicate that NPC-related and cholesterol perturbation-induced axonal pathology is associated with an abnormal signaling pathway consisting in p38 MAPK activation leading to Mdm2-mediated p53 degradation, followed by ROCK activation. These results also suggest new targets for pharmacological treatment of NPC disease and other diseases associated with disruption of cholesterol metabolism.

  15. Modeling molecular mechanisms in the axon (United States)

    de Rooij, R.; Miller, K.E.; Kuhl, E.


    Axons are living systems that display highly dynamic changes in stiffness, viscosity, and internal stress. However, the mechanistic origin of these phenomenological properties remains elusive. Here we establish a computational mechanics model that interprets cellular-level characteristics as emergent properties from molecular-level events. We create an axon model of discrete microtubules, which are connected to neighboring microtubules via discrete crosslinking mechanisms that obey a set of simple rules. We explore two types of mechanisms: passive and active crosslinking. Our passive and active simulations suggest that the stiffness and viscosity of the axon increase linearly with the crosslink density, and that both are highly sensitive to the crosslink detachment and reattachment times. Our model explains how active crosslinking with dynein motors generates internal stresses and actively drives axon elongation. We anticipate that our model will allow us to probe a wide variety of molecular phenomena–both in isolation and in interaction–to explore emergent cellular-level features under physiological and pathological conditions. PMID:28603326

  16. Macrophages Promote Axon Regeneration with Concurrent Neurotoxicity

    NARCIS (Netherlands)

    Gensel, J.C.; Nakamura, S.; Guan, Z.; Rooijen, van N.; Ankeny, D.P.; Popovich, P.G.


    Activated macrophages can promote regeneration of CNS axons. However, macrophages also release factors that kill neurons. These opposing functions are likely induced simultaneously but are rarely considered together in the same experimental preparation. A goal of this study was to unequivocally

  17. Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration. (United States)

    Kaplan, Andrew; Morquette, Barbara; Kroner, Antje; Leong, SooYuen; Madwar, Carolin; Sanz, Ricardo; Banerjee, Sara L; Antel, Jack; Bisson, Nicolas; David, Samuel; Fournier, Alyson E


    Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Specific effects of c-Jun NH2-terminal kinase-interacting protein 1 in neuronal axons

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    Shu Tang


    Full Text Available c-Jun NH2-terminal kinase (JNK-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B (TrkB anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of TrkB anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed TrkB complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of TrkB gradually increased in axon terminals. However, the distribution of TrkB reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of TrkB after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of TrkB in dendrites. These findings confirm that JNK-interacting protein 1 can interact with TrkB in neuronal cells, and can regulate the transport of TrkB in axons, but not in dendrites.

  19. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance. (United States)

    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E


    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  20. Mitochondria Localize to Injured Axons to Support Regeneration. (United States)

    Han, Sung Min; Baig, Huma S; Hammarlund, Marc


    Axon regeneration is essential to restore the nervous system after axon injury. However, the neuronal cell biology that underlies axon regeneration is incompletely understood. Here we use in vivo, single-neuron analysis to investigate the relationship between nerve injury, mitochondrial localization, and axon regeneration. Mitochondria translocate into injured axons so that average mitochondria density increases after injury. Moreover, single-neuron analysis reveals that axons that fail to increase mitochondria have poor regeneration. Experimental alterations to axonal mitochondrial distribution or mitochondrial respiratory chain function result in corresponding changes to regeneration outcomes. Axonal mitochondria are specifically required for growth-cone migration, identifying a key energy challenge for injured neurons. Finally, mitochondrial localization to the axon after injury is regulated in part by dual-leucine zipper kinase 1 (DLK-1), a conserved regulator of axon regeneration. These data identify regulation of axonal mitochondria as a new cell-biological mechanism that helps determine the regenerative response of injured neurons. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Axon degeneration: make the Schwann cell great again

    Directory of Open Access Journals (Sweden)

    Keit Men Wong


    Full Text Available Axonal degeneration is a pivotal feature of many neurodegenerative conditions and substantially accounts for neurological morbidity. A widely used experimental model to study the mechanisms of axonal degeneration is Wallerian degeneration (WD, which occurs after acute axonal injury. In the peripheral nervous system (PNS, WD is characterized by swift dismantling and clearance of injured axons with their myelin sheaths. This is a prerequisite for successful axonal regeneration. In the central nervous system (CNS, WD is much slower, which significantly contributes to failed axonal regeneration. Although it is well-documented that Schwann cells (SCs have a critical role in the regenerative potential of the PNS, to date we have only scarce knowledge as to how SCs 'sense' axonal injury and immediately respond to it. In this regard, it remains unknown as to whether SCs play the role of a passive bystander or an active director during the execution of the highly orchestrated disintegration program of axons. Older reports, together with more recent studies, suggest that SCs mount dynamic injury responses minutes after axonal injury, long before axonal breakdown occurs. The swift SC response to axonal injury could play either a pro-degenerative role, or alternatively a supportive role, to the integrity of distressed axons that have not yet committed to degenerate. Indeed, supporting the latter concept, recent findings in a chronic PNS neurodegeneration model indicate that deactivation of a key molecule promoting SC injury responses exacerbates axonal loss. If this holds true in a broader spectrum of conditions, it may provide the grounds for the development of new glia-centric therapeutic approaches to counteract axonal loss.

  2. Transportation

    International Nuclear Information System (INIS)



    Here is the decree of the thirtieth of July 1998 relative to road transportation, to trade and brokerage of wastes. It requires to firms which carry out a road transportation as well as to traders and to brokers of wastes to declare their operations to the prefect. The declaration has to be renewed every five years. (O.M.)

  3. Imaging of mitochondrial dynamics in motor and sensory axons of living mice. (United States)

    Bolea, Irene; Gan, Wen-Biao; Manfedi, Giovanni; Magrané, Jordi


    Appropriate distribution and supply of mitochondria to critical neuronal sites are thought to be necessary for the normal maintenance of neuronal architecture and activity, including synaptic plasticity and function. Imaging of neurons in vitro has provided understanding of the basic mechanisms of mitochondrial transport and the regulation of mitochondrial dynamics. However, in vivo imaging studies of neurons are preferable to in vitro approaches because of the advantage of being performed in their natural environment. Here, we present useful protocols to image and study axonal transport of mitochondria in vivo, in the peripheral nerves of mice. Imaging in motor and sensory axons of living mice allows researchers to analyze mitochondrial dynamics in two distinct neuronal populations that are often affected in peripheral neuropathies.

  4. Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

    Directory of Open Access Journals (Sweden)

    Sandrine Joly


    Full Text Available The lack of axonal regeneration and neuronal cell death causes permanent neurological deficits in the injured CNS. Using the classical CNS injury model of optic nerve crush in mice, ciliary neurotrophic factor (CNTF was found to stimulate retinal ganglion cell (RGC survival and axonal growth, but in an incomplete fashion. The elucidation of molecular mechanisms impairing CNTF-induced axonal regeneration is paramount to promote visual recovery. In the present study, we sought to evaluate the contribution of sphingosine 1-phosphate receptor 1 (S1PR1 to the neuroprotective and regenerative effects of CNTF. The transduction of retinal cells with adeno-associated viruses (AAV allowed to activate CNTF/signal transducer and activator of transcription 3 (Stat3 signaling and to modulate S1PR1 expression in RGCs. Our results showed that CNTF/Stat3 prevented injury-induced S1PR1 downregulation. Silencing S1PR1 in RGCs significantly enhanced CNTF-induced axonal growth in the injured optic nerve. In contrast, RGC survival was markedly decreased when S1PR1 was repressed with viral vectors. The level of phosphorylated Stat3 (P-Stat3, an intracellular mediator of CNTF, did not fluctuate after S1PR1 inhibition and CNTF stimulation. Collectively, these results suggest that S1PR1 acts as a major regulator of retinal neuron survival and restricts the RGC growth response induced by CNTF.

  5. Motor axon excitability during Wallerian degeneration

    DEFF Research Database (Denmark)

    Moldovan, Mihai; Alvarez, Susana; Krarup, Christian


    Axonal loss and degeneration are major factors in determining long-term outcome in patients with peripheral nerve disorders or injury. Following loss of axonal continuity, the isolated nerve stump distal to the lesion undergoes Wallerian degeneration in several phases. In the initial 'latent' phase...... at ankle distal to axotomy were monitored by 'threshold-tracking'. The plantar compound muscle action potentials (CMAPs) were recorded under anesthesia in three animal models: 8-week-old wild-type mice, 8-week-old slow Wallerian degeneration mutant mice and 3-year-old cats. We found that the progressive...... decrease in CMAP following crush injury was associated with slowing of conduction and marked abnormalities in excitability: increased peak threshold deviations during both depolarizing and hyperpolarizing threshold electrotonus, enhanced superexcitability during the recovery cycle and increased rheobase...

  6. B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS (United States)

    O’Donovan, Kevin J.; Ma, Kaijie; Guo, Hengchang; Wang, Chen; Sun, Fang; Han, Seung Baek; Kim, Hyukmin; Wong, Jamie K.; Charron, Jean; Zou, Hongyan; Son, Young-Jin; He, Zhigang


    Activation of intrinsic growth programs that promote developmental axon growth may also facilitate axon regeneration in injured adult neurons. Here, we demonstrate that conditional activation of B-RAF kinase alone in mouse embryonic neurons is sufficient to drive the growth of long-range peripheral sensory axon projections in vivo in the absence of upstream neurotrophin signaling. We further show that activated B-RAF signaling enables robust regenerative growth of sensory axons into the spinal cord after a dorsal root crush as well as substantial axon regrowth in the crush-lesioned optic nerve. Finally, the combination of B-RAF gain-of-function and PTEN loss-of-function promotes optic nerve axon extension beyond what would be predicted for a simple additive effect. We conclude that cell-intrinsic RAF signaling is a crucial pathway promoting developmental and regenerative axon growth in the peripheral and central nervous systems. PMID:24733831

  7. Quantitative analysis of microtubule transport in growing nerve processes

    DEFF Research Database (Denmark)

    Ma*, Ytao; Shakiryanova*, Dinara; Vardya, Irina


    assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured...

  8. Sensory axonal dysfunction in cervical radiculopathy. (United States)

    Sung, Jia-Ying; Tani, Jowy; Hung, Kuo-Sheng; Lui, Tai-Ngar; Lin, Cindy Shin-Yi


    The aim of this study was to evaluate changes in sensory axonal excitability in the distal nerve in patients with cervical radiculopathy. The patients were classified by the findings of cervical MRI into two subgroups: 22 patients with C6/7 root compression and 25 patients with cervical cord and root compression above/at C6/7. Patients were investigated using conventional nerve conduction studies (NCS) and nerve excitability testing. Sensory nerve excitability testing was undertaken with stimulation at the wrist and recording from digit II (dermatome C6/7). The results were compared with healthy controls. Both preoperative and postoperative tests were performed if the patient underwent surgery. Sensory axonal excitability was significantly different in both cohorts compared with healthy controls, including prolonged strength-duration time constant, reduced S2 accommodation, increased threshold electrotonus hyperpolarisation (TEh (90-100 ms)), and increased superexcitability. The changes in these excitability indices are compatible with axonal membrane hyperpolarisation. In five patients who underwent surgery, the postoperative sensory excitability was tested after 1 week, and showed significant changes in TE (TEh (90-100 ms) and TEh slope, pcervical radiculopathy. These findings suggest that the hyperpolarised pattern might be due to Na(+)-K(+) ATPase overactivation induced by proximal ischaemia, or could reflect the remyelinating process. Distal sensory axons were hyperpolarised even though there were no changes in NCS, suggesting that nerve excitability testing may be more sensitive to clinical symptoms than NCS in patients with cervical radiculopathy. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to

  9. Unidirectional ephaptic stimulation between two myelinated axons. (United States)

    Capllonch-Juan, Miguel; Kolbl, Florian; Sepulveda, Francisco


    Providing realistic sensory feedback for prosthetic devices strongly relies on an accurate modelling of machine-nerve interfaces. Models of these interfaces in the peripheral nervous system usually neglect the effects that ephaptic coupling can have on the selectivity of stimulating electrodes. In this contribution, we study the ephaptic stimulation between myelinated axons and show its relation with the separation between fibers and the conductivity of the medium that surrounds them.

  10. Multifunctional Silk Nerve Guides for Axon Outgrowth (United States)

    Tupaj, Marie C.

    Peripheral nerve regeneration is a critical issue as 2.8% of trauma patients present with this type of injury, estimating a total of 200,000 nerve repair procedures yearly in the United States. While the peripheral nervous system exhibits slow regeneration, at a rate of 0.5 mm -- 9 mm/day following trauma, this regenerative ability is only possible under certain conditions. Clinical repairs have changed slightly in the last 30 years and standard methods of treatment include suturing damaged nerve ends, allografting, and autografting, with the autograft the gold standard of these approaches. Unfortunately, the use of autografts requires a second surgery and there is a shortage of nerves available for grafting. Allografts are a second option however allografts have lower success rates and are accompanied by the need of immunosuppressant drugs. Recently there has been a focus on developing nerve guides as an "off the shelf" approach. Although some natural and synthetic guidance channels have been approved by the FDA, these nerve guides are unfunctionalized and repair only short gaps, less than 3 cm in length. The goal of this project was to identify strategies for functionalizing peripheral nerve conduits for the outgrowth of neuron axons in vitro . To accomplish this, two strategies (bioelectrical and biophysical) were indentified for increasing axon outgrowth and promoting axon guidance. Bioelectrical strategies exploited electrical stimulation for increasing neurite outgrowth. Biophysical strategies tested a range of surface topographies for axon guidance. Novel methods were developed for integrating electrical and biophysical strategies into silk films in 2D. Finally, a functionalized nerve conduit system was developed that integrated all strategies for the purpose of attaching, elongating, and guiding nervous tissue in vitro. Future directions of this work include silk conduit translation into a rat sciatic nerve model in vivo for the purpose of repairing long

  11. Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling inDrosophilaaxon guidance. (United States)

    Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward


    Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

  12. Axonal branching patterns of nucleus accumbens neurons in the rat. (United States)

    Tripathi, Anushree; Prensa, Lucía; Cebrián, Carolina; Mengual, Elisa


    The patterns of axonal collateralization of nucleus accumbens (Acb) projection neurons were investigated in the rat by means of single-axon tracing techniques using the anterograde tracer biotinylated dextran amine. Seventy-three axons were fully traced, originating from either the core (AcbC) or shell (AcbSh) compartment, as assessed by differential calbindin D28k-immunoreactivity. Axons from AcbC and AcbSh showed a substantial segregation in their targets; target areas were either exclusively or preferentially innervated from AcbC or AcbSh. Axon collaterals in the subthalamic nucleus were found at higher than expected frequencies; moreover, these originated exclusively in the dorsal AcbC. Intercompartmental collaterals were observed from ventral AcbC axons into AcbSh, and likewise, interconnections at pallidal and mesencephalic levels were also observed, although mostly from AcbC axons toward AcbSh targets, possibly supporting crosstalk between the two subcircuits at several levels. Cell somata giving rise to short-range accumbal axons, projecting to the ventral pallidum (VP), were spatially intermingled with others, giving rise to long-range axons that innervated VP and more caudal targets. This anatomical organization parallels that of the dorsal striatum and provides the basis for possible dual direct and indirect actions from a single axon on either individual or small sets of neurons. Copyright © 2010 Wiley-Liss, Inc.

  13. Transportation

    National Research Council Canada - National Science Library

    Allshouse, Michael; Armstrong, Frederick Henry; Burns, Stephen; Courts, Michael; Denn, Douglas; Fortunato, Paul; Gettings, Daniel; Hansen, David; Hoffman, Douglas; Jones, Robert


    .... The ability of the global transportation industry to rapidly move passengers and products from one corner of the globe to another continues to amaze even those wise to the dynamics of such operations...

  14. Trafficking of Kv2.1 Channels to the Axon Initial Segment by a Novel Nonconventional Secretory Pathway

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Watanabe, Shoji; Stas, Jeroen Ingrid


    the localization of Kv2.1 in these two different membrane compartments in cultured rat hippocampal neurons of mixed sex. Our data uncover a unique ability of Kv2.1 channels to use two molecularly distinct trafficking pathways to accomplish this. Somatodendritic Kv2.1 channels are targeted by the conventional...... secretory pathway, whereas axonal Kv2.1 channels are targeted by a nonconventional trafficking pathway independent of the Golgi apparatus. We further identified a new AIS trafficking motif in the C-terminus of Kv2.1, and show that putative phosphorylation sites in this region are critical for the restricted.......SIGNIFICANCE STATEMENT Our study uncovered a novel mechanism that targets the Kv2.1 voltage-gated potassium channel to two distinct trafficking pathways and two distinct subcellular destinations: the somatodendritic plasma membrane and that of the axon initial segment. We also identified a distinct motif, including...

  15. Regulation and dysregulation of axon infrastructure by myelinating glia. (United States)

    Pan, Simon; Chan, Jonah R


    Axon loss and neurodegeneration constitute clinically debilitating sequelae in demyelinating diseases such as multiple sclerosis, but the underlying mechanisms of secondary degeneration are not well understood. Myelinating glia play a fundamental role in promoting the maturation of the axon cytoskeleton, regulating axon trafficking parameters, and imposing architectural rearrangements such as the nodes of Ranvier and their associated molecular domains. In the setting of demyelination, these changes may be reversed or persist as maladaptive features, leading to axon degeneration. In this review, we consider recent insights into axon-glial interactions during development and disease to propose that disruption of the cytoskeleton, nodal architecture, and other components of axon infrastructure is a potential mediator of pathophysiological damage after demyelination. © 2017 Pan and Chan.

  16. Active polysomes in the axoplasm of the squid giant axon. (United States)

    Giuditta, A; Menichini, E; Perrone Capano, C; Langella, M; Martin, R; Castigli, E; Kaplan, B B


    Axons and axon terminals are widely believed to lack the capacity to synthesize proteins, relying instead on the delivery of proteins made in the perikaryon. In agreement with this view, axoplasmic proteins synthesized by the isolated giant axon of the squid are believed to derive entirely from periaxonal glial cells. However, squid axoplasm is known to contain the requisite components of an extra-mitochondrial protein synthetic system, including protein factors, tRNAs, rRNAs, and a heterogeneous family of mRNAs. Hence, the giant axon could, in principle, maintain an endogenous protein synthetic capacity. Here, we report that the squid giant axon also contains active polysomes and mRNA, which hybridizes to a riboprobe encoding murine neurofilament protein. Taken together, these findings provide direct evidence that proteins (including the putative neuron-specific neurofilament protein) are also synthesized de novo in the axonal compartment.

  17. Axon-glia interaction and membrane traffic in myelin formation


    White, Robin; Krämer-Albers, Eva-Maria


    In vertebrate nervous systems myelination of neuronal axons has evolved to increase conduction velocity of electrical impulses with minimal space and energy requirements. Myelin is formed by specialized glial cells which ensheath axons with a lipid-rich insulating membrane. Myelination is a multi-step process initiated by axon-glia recognition triggering glial polarization followed by targeted myelin membrane expansion and compaction. Thereby, a myelin sheath of complex subdomain structure is...

  18. Antioxidants Halt Axonal Degeneration in a Mouse Model of X-Adrenoleukodystrophy (United States)

    López-Erauskin, Jone; Fourcade, Stéphane; Galino, Jorge; Ruiz, Montserrat; Schlüter, Agatha; Naudi, Alba; Jove, Mariona; Portero-Otin, Manuel; Pamplona, Reinald; Ferrer, Isidre; Pujol, Aurora


    Objective Axonal degeneration is a main contributor to disability in progressive neurodegenerative diseases in which oxidative stress is often identified as a pathogenic factor. We aim to demonstrate that antioxidants are able to improve axonal degeneration and locomotor deficits in a mouse model of X-adrenoleukodystrophy (X-ALD). Methods X-ALD is a lethal disease caused by loss of function of the ABCD1 peroxisomal transporter of very long chain fatty acids (VLCFA). The mouse model for X-ALD exhibits a late onset neurological phenotype with locomotor disability and axonal degeneration in spinal cord resembling the most common phenotype of the disease, adrenomyeloneuropathy (X-AMN). Recently, we identified oxidative damage as an early event in life, and the excess of VLCFA as a generator of radical oxygen species (ROS) and oxidative damage to proteins in X-ALD. Results Here, we prove the capability of the antioxidants N-acetyl-cysteine, α-lipoic acid, and α-tocopherol to scavenge VLCFA-dependent ROS generation in vitro. Furthermore, in a preclinical setting, the cocktail of the 3 compounds reversed: (1) oxidative stress and lesions to proteins, (2) immunohistological signs of axonal degeneration, and (3) locomotor impairment in bar cross and treadmill tests. Interpretation We have established a direct link between oxidative stress and axonal damage in a mouse model of neurodegenerative disease. This conceptual proof of oxidative stress as a major disease-driving factor in X-AMN warrants translation into clinical trials for X-AMN, and invites assessment of antioxidant strategies in axonopathies in which oxidative damage might be a contributing factor. Ann Neurol 2011; PMID:21786300

  19. Presynaptic localization of Smn and hnRNP R in axon terminals of embryonic and postnatal mouse motoneurons.

    Directory of Open Access Journals (Sweden)

    Benjamin Dombert

    Full Text Available Spinal muscular atrophy (SMA is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

  20. Glycogen phosphorylation and Lafora disease. (United States)

    Roach, Peter J


    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. A new model of traumatic axonal injury to determine the effects of strain and displacement rates. (United States)

    Singh, Anita; Lu, Ying; Chen, Chaoyang; Kallakuri, Srinivasu; Cavanaugh, John M


    Traumatic brain injury (TBI) continues to be a major health problem, with over 500,000 cases per year with a societal cost of approximately $85 billion in the US. Motor vehicle accidents are the leading cause of such injuries. In many cases of TBI widespread disruption of the axons occurs through a process known as diffuse axonal injury (DAI) or traumatic axonal injury (TAI). In the current study, an in vivo TAI model was developed using spinal nerve roots of adult rats. This model was used to determine functional and structural responses of axons to various strains and displacement rates. Fifty-six L5 dorsal nerve roots were each subjected to a predetermined strain range (20%) at a specified displacement rate (0.01 mm/sec and 15 mm/sec) only once. Image analysis was used to determine actual strains on the roots during the pull. Neurophysiologic recordings were performed on the nerve root before and after stretch to determine functional changes in response to stretch, including conduction velocity (CV) and area of the evoked compound action potential (CAP). Structural changes including vascular injury, axotomy, and impaired axoplasmic transport (IAT) were evaluated using hematoxylin and eosin, Palmgren silver impregnation and beta-APP staining techniques, respectively. Results showed that CV and the area of the CAP decreased as strain and displacement rate increased. Also, threshold strains for complete nerve conduction loss were 16% and 9% at 0.01 mm/sec and 15 mm/sec rate, respectively. These threshold values indicate the rate dependency of functional injury and indicate that axons tolerate slow loading rates better than higher loading rates. Histological studies revealed increased spacing, tearing of axons, IAT and occurrence of hemorrhage to be strain and displacement rate dependent. Linear relationships existed between the increasing strain and the occurrence rate of axonal injury as evidenced by multiple indicators (IAT, hemorrhage, torn fibers or primary

  2. Origin, course, and laterality of spinocerebellar axons in the North American opossum, Didelphis virginiana. (United States)

    Terman, J R; Wang, X M; Martin, G F


    Spinocerebellar axons have been studied extensively in placental mammals, but there have been no full reports on their origin, laterality, or spinal course in any marsupial. We have used the North American opossum (Didelphis virginiana) to obtain such information and to ask whether any spinocerebellar neurons innervate both the anterior and posterior lobes of the cerebellum through axonal collaterals. To identify spinal neurons that project to the cerebellum, we employed the retrograde transport of Fluoro-Gold (FG) from the anterior lobe, the main target of spinocerebellar axons. In some cases, cerebellar injections of FG were combined with hemisections of the rostral cervical or midthoracic spinal cord, so that laterality of spinocerebellar connections could be established. To determine whether single neurons project to both the anterior lobe and the posterior lobe, injections of Fast Blue (FB) into the anterior lobe were combined with injections of Diamidino yellow (DY) or rhodamine B dextran (RBD) into the posterior lobe, or vice versa. Following injections of FG into the anterior lobe, neurons were labeled throughout the length of the spinal cord, which differed in laminar distribution and laterality of their projections. Among other areas, neurons were labeled in the central cervical nucleus, the nucleus centrobasalis, Clarke's nucleus, the dorsal horn dorsal spinocerebellar tract area, the spinal border region, and Stilling's nucleus. When anterior lobe injections of FB were combined with injections of RBD or DY into the posterior lobe, or vice versa, some double-labeled neurons were present in all major spinocerebellar groups. Cerebellar injections of FG also retrogradely labeled spinocerebellar axons, allowing us to document their locations in the gray matter as well as within the periphery of the lateral and ventral funiculi at all spinal levels. A few spinocerebellar axons also were found in the dorsal funiculus (a dorsal column-spinocerebellar tract

  3. Schwann Cell Glycogen Selectively Supports Myelinated Axon Function (United States)

    Brown, Angus M; Evans, Richard D; Black, Joel; Ransom, Bruce R


    Objectives Interruption of energy supply to peripheral axons is a cause of axon loss. We determined if glycogen was present in mammalian peripheral nerve, and if it supported axon conduction during aglycemia. Methods We used biochemical assay and electron microscopy to determine the presence of glycogen, and electrophysiology to monitor axon function. Results Glycogen was present in sciatic nerve, its concentration varying directly with ambient [glucose]. Electron microscopy detected glycogen granules primarily in myelinating Schwann cell cytoplasm and these diminished after exposure to aglycemia. During aglycemia, conduction failure in large myelinated axons (A fibers) mirrored the time-course of glycogen loss. Latency to CAP failure was directly related to nerve glycogen content at aglycemia onset. Glycogen did not benefit the function of slow-conducting, small diameter unmyelinated axons (C fibers) during aglycemia. Blocking glycogen breakdown pharmacologically accelerated CAP failure during aglycemia in A fibers, but not in C fibers. Lactate was as effective as glucose in supporting sciatic nerve function, and was continuously released into the extracellular space in the presence of glucose and fell rapidly during aglycemia. Interpretation Our findings indicated that glycogen is present in peripheral nerve, primarily in myelinating Schwann cells, and exclusively supports large diameter, myelinated axon conduction during aglycemia. Available evidence suggests that peripheral nerve glycogen breaks down during aglycemia and is passed, probably as lactate, to myelinated axons to support function. Unmyelinated axons are not protected by glycogen and are more vulnerable to dysfunction during periods of hypoglycemia. PMID:23034913

  4. Axonal branching patterns of ventral pallidal neurons in the rat. (United States)

    Tripathi, Anushree; Prensa, Lucía; Mengual, Elisa


    The ventral pallidum (VP) is a key component of the cortico-basal ganglia circuits that process motivational and emotional information, and also a crucial site for reward. Although the main targets of the two VP compartments, medial (VPm) and lateral (VPl) have already been established, the collateralization patterns of individual axons have not previously been investigated. Here we have fully traced eighty-four axons from VPm, VPl and the rostral extension of VP into the olfactory tubercle (VPr), using the anterograde tracer biotinylated dextran amine in the rat. Thirty to fifty percent of axons originating from VPm and VPr collateralized in the mediodorsal thalamic nucleus and lateral habenula, indicating a close association between the ventral basal ganglia-thalamo-cortical loop and the reward network at the single axon level. Additional collateralization of these axons in diverse components of the extended amygdala and corticopetal system supports a multisystem integration that may take place at the basal forebrain. Remarkably, we did not find evidence for a sharp segregation in the targets of axons arising from the two VP compartments, as VPl axons frequently collateralized in the caudal lateral hypothalamus and ventral tegmental area, the well-known targets of VPm, while VPm axons, in turn, also collateralized in typical VPl targets such as the subthalamic nucleus, substantia nigra pars compacta and reticulata, and retrorubral field. Nevertheless, VPl and VPm displayed collateralization patterns that paralleled those of dorsal pallidal components, confirming at the single axon level the parallel organization of functionally different basal ganglia loops.

  5. Differences in excitability properties of FDI and ADM motor axons. (United States)

    Bae, Jong Seok; Sawai, Setsu; Misawa, Sonoko; Kanai, Kazuaki; Isose, Sagiri; Kuwabara, Satoshi


    The first dorsal interosseous (FDI) and abductor digiti minimi (ADM) muscles are innervated by the same ulnar nerve, but studies have shown that the former is much more severely affected in amyotrophic lateral sclerosis. In this study, threshold tracking was used to investigate whether membrane properties differ between FDI and ADM motor axons. In 12 normal subjects, compound muscle action potentials were recorded from FDI and ADM after ulnar nerve stimulation at the wrist. The strength-duration time constant was significantly longer in the FDI axons than in the ADM axons, and latent addition studies showed greater threshold changes at the conditioning-test stimulus of 0.2 ms in FDI than in ADM axons. These findings suggest that nodal persistent sodium conductances are more prominent in FDI axons than in ADM axons, and therefore excitability is physiologically higher in FDI axons. Even in the same nerve at the same sites, membrane properties of FDI and ADM motor axons differ significantly, and thus their axonal/neuronal responses to disease may also differ.

  6. Axon Regeneration Is Regulated by Ets-C/EBP Transcription Complexes Generated by Activation of the cAMP/Ca2+ Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Chun Li


    Full Text Available The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. In Caenorhabditis elegans, the JNK and p38 MAPK pathways are important for axon regeneration. Axonal injury induces expression of the svh-2 gene encoding a receptor tyrosine kinase, stimulation of which by the SVH-1 growth factor leads to activation of the JNK pathway. Here, we identify ETS-4 and CEBP-1, related to mammalian Ets and C/EBP, respectively, as transcriptional activators of svh-2 expression following axon injury. ETS-4 and CEBP-1 function downstream of the cAMP and Ca2+-p38 MAPK pathways, respectively. We show that PKA-dependent phosphorylation of ETS-4 promotes its complex formation with CEBP-1. Furthermore, activation of both cAMP and Ca2+ signaling is required for activation of svh-2 expression. Thus, the cAMP/Ca2+ signaling pathways cooperatively activate the JNK pathway, which then promotes axon regeneration.

  7. Propofol directly increases tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Robert A Whittington


    Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

  8. Death Receptor 6 Promotes Wallerian Degeneration in Peripheral Axons. (United States)

    Gamage, Kanchana K; Cheng, Irene; Park, Rachel E; Karim, Mardeen S; Edamura, Kazusa; Hughes, Christopher; Spano, Anthony J; Erisir, Alev; Deppmann, Christopher D


    Axon degeneration during development is required to sculpt a functional nervous system and is also a hallmark of pathological insult, such as injury [1, 2]. Despite similar morphological characteristics, very little overlap in molecular mechanisms has been reported between pathological and developmental degeneration [3-5]. In the peripheral nervous system (PNS), developmental axon pruning relies on receptor-mediated extrinsic degeneration mechanisms to determine which axons are maintained or degenerated [5-7]. Receptors have not been implicated in Wallerian axon degeneration; instead, axon autonomous, intrinsic mechanisms are thought to be the primary driver for this type of axon disintegration [8-10]. Here we survey the role of neuronally expressed, paralogous tumor necrosis factor receptor super family (TNFRSF) members in Wallerian degeneration. We find that an orphan receptor, death receptor 6 (DR6), is required to drive axon degeneration after axotomy in sympathetic and sensory neurons cultured in microfluidic devices. We sought to validate these in vitro findings in vivo using a transected sciatic nerve model. Consistent with the in vitro findings, DR6 -/- animals displayed preserved axons up to 4 weeks after injury. In contrast to phenotypes observed in Wld s and Sarm1 -/- mice, preserved axons in DR6 -/- animals display profound myelin remodeling. This indicates that deterioration of axons and myelin after axotomy are mechanistically distinct processes. Finally, we find that JNK signaling after injury requires DR6, suggesting a link between this novel extrinsic pathway and the axon autonomous, intrinsic pathways that have become established for Wallerian degeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.


    DEFF Research Database (Denmark)

    Fog, Jacob; Khoshbouei, H; Holy, M

    The dopamine transporter(DAT) plays a key role in clearing extracellular dopamine(DA) from the synapse. Moreover DAT is a target for the action of widely abused psychostimulants such as cocaine and amphetamine(AMPH). AMPH is a substrate for the DAT and promotes the reversal of transport and thus...

  10. Rare autism-associated variants implicate syntaxin 1 (STX1 R26Q) phosphorylation and the dopamine transporter (hDAT R51W) in dopamine neurotransmission and behaviors

    DEFF Research Database (Denmark)

    Cartier, Etienne; Hamilton, Peter J; Belovich, Andrea N


    BACKGROUND: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through...

  11. Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons. (United States)

    Niwa, Shinsuke


    Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

  12. Modelling the Krebs cycle and oxidative phosphorylation. (United States)

    Korla, Kalyani; Mitra, Chanchal K


    The Krebs cycle and oxidative phosphorylation are the two most important sets of reactions in a eukaryotic cell that meet the major part of the total energy demands of a cell. In this paper, we present a computer simulation of the coupled reactions using open source tools for simulation. We also show that it is possible to model the Krebs cycle with a simple black box with a few inputs and outputs. However, the kinetics of the internal processes has been modelled using numerical tools. We also show that the Krebs cycle and oxidative phosphorylation together can be combined in a similar fashion - a black box with a few inputs and outputs. The Octave script is flexible and customisable for any chosen set-up for this model. In several cases, we had no explicit idea of the underlying reaction mechanism and the rate determining steps involved, and we have used the stoichiometric equations that can be easily changed as and when more detailed information is obtained. The script includes the feedback regulation of the various enzymes of the Krebs cycle. For the electron transport chain, the pH gradient across the membrane is an essential regulator of the kinetics and this has been modelled empirically but fully consistent with experimental results. The initial conditions can be very easily changed and the simulation is potentially very useful in a number of cases of clinical importance.

  13. Tyrosine phosphorylation in human lymphomas

    NARCIS (Netherlands)

    Haralambieva, E; Jones, M.; Roncador, GM; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, KM; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, PM; Pulford, K; Mason, DY


    In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated


    Energy Technology Data Exchange (ETDEWEB)



    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  15. Enkephalins affect hippocampal membrane phosphorylation

    NARCIS (Netherlands)

    Bär, P.R; Schotman, P.; Gispen, W.H.


    Slices of rat brain hippocampus were incubated with methionine-enkephalin, leucine-enkephalin, [Des-Tyr1] methionine-enkephalin or etorphin. After incubation the endogenous phosphorylation of proteins was measured using crude mitochhondrial fractions prepared from the incubated slices. Methione- and

  16. Is action potential threshold lowest in the axon?

    NARCIS (Netherlands)

    Kole, Maarten H. P.; Stuart, Greg J.


    Action potential threshold is thought to be lowest in the axon, but when measured using conventional techniques, we found that action potential voltage threshold of rat cortical pyramidal neurons was higher in the axon than at other neuronal locations. In contrast, both current threshold and voltage

  17. Wnts guide longitudinal axon tracts in the brain

    NARCIS (Netherlands)

    Prasad, A.A.


    The human brain contains more than 10 billion neurons that form over 10 trillion connections. The establishment of these connections during development requires axons to extend through the extracellular environment to their synaptic targets. This process of axon guidance is mediated by molecular

  18. Nutritional tryptophan restriction impairs plasticity of retinotectal axons during the critical period. (United States)

    Penedo, Letícia Abel; Oliveira-Silva, Priscilla; Gonzalez, Ericka M C; Maciel, Rafaela; Jurgilas, Patricia B; Melibeu, Adriana da Cunha Faria; Campello-Costa, Paula; Serfaty, Claudio Alberto


    The use-dependent specification of neural circuits occurs during post-natal development with a conspicuous influence of environmental factors, such as malnutrition that interferes with the major steps of brain maturation. Serotonin (5-HT), derived exclusively from the essential aminoacid tryptophan, is involved in mechanisms of development and use-dependent plasticity of the central nervous system. We studied the effects of the nutritional restriction of tryptophan in the plasticity of uncrossed retinotectal axons following a retinal lesion to the contralateral retina during the critical period in pigmented rats. Litters were fed through their mothers with a low tryptophan content diet, based on corn and gelatin, a complemented diet with standard tryptophan requirements for rodents or standard laboratory diet. The results suggest a marked reduction in the plasticity of intact axons into denervated territories in the tryptophan restricted group in comparison to control groups. Tryptophan complementation between PND10-21 completely restored retinotectal plasticity. However, the re-introduction of tryptophan after the end of the critical period (between PND28-P41) did not restore the sprouting ability of uncrossed axons suggesting a time-dependent effect to the reversion of plasticity deficits. Tryptophan-restricted animals showed a reduced activity of matrix metalloproteinase-9 and altered expressions of phosphorylated forms of ERK1/2 and AKT. Our results demonstrate the influence of this essential aminoacid as a modulator of neural plasticity during the critical period through the reduction of serotonin content which alters plasticity-related signaling pathways and matrix degradation.

  19. SnoN facilitates axonal regeneration after spinal cord injury.

    Directory of Open Access Journals (Sweden)

    Jiun L Do

    Full Text Available Adult CNS neurons exhibit a reduced capacity for growth compared to developing neurons, due in part to downregulation of growth-associated genes as development is completed. We tested the hypothesis that SnoN, an embryonically regulated transcription factor that specifies growth of the axonal compartment, can enhance growth in injured adult neurons. In vitro, SnoN overexpression in dissociated adult DRG neuronal cultures significantly enhanced neurite outgrowth. Moreover, TGF-β1, a negative regulator of SnoN, inhibited neurite outgrowth, and SnoN over-expression overcame this inhibition. We then examined whether SnoN influenced axonal regeneration in vivo: indeed, expression of a mutant form of SnoN resistant to degradation significantly enhanced axonal regeneration following cervical spinal cord injury, despite peri-lesional upregulation of TGF-β1. Thus, a developmental mechanism that specifies extension of the axonal compartment also promotes axonal regeneration after adult CNS injury.

  20. Motor Axonal Regeneration After Partial and Complete Spinal Cord Transection (United States)

    Lu, Paul; Blesch, Armin; Graham, Lori; Wang, Yaozhi; Samara, Ramsey; Banos, Karla; Haringer, Verena; Havton, Leif; Weishaupt, Nina; Bennett, David; Fouad, Karim; Tuszynski, Mark H.


    We subjected rats to either partial mid-cervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor (BDNF) gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared to several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair, and the need for additional control and shaping of axonal regeneration. PMID:22699902

  1. Plasticity of the Axon Initial Segment

    DEFF Research Database (Denmark)

    Petersen, Anders Victor; Cotel, Florence; Perrier, Jean François


    undergo important modifications during development. The development of the AIS is governed by intrinsic mechanisms. In addition, surrounding neuronal networks modify its maturation. As a result, neurons get tuned to particular physiological functions. Neuronal activity also influences the morphology......The axon initial segment (AIS) is a key neuronal compartment because it is responsible for action potential initiation. The local density of Na+ channels, the biophysical properties of K+ channels, as well as the length and diameter of the AIS determine the spiking of neurons. These parameters...... of the mature AIS. When excitatory neurons are hyperactive, their AIS undergo structural changes that decrease their excitability and thereby maintain the activity within a given range. These slow homeostatic regulatory mechanisms occur on a time scale of hours or days. In contrast, the activation...

  2. Prolyl Isomerase Pin1 Regulates Axon Guidance by Stabilizing CRMP2A Selectively in Distal Axons

    Czech Academy of Sciences Publication Activity Database

    Balaštík, Martin; Zhou, X.Z.; Alberich-Jorda, Meritxell; Weissová, Romana; Žiak, Jakub; Pazyra-Murphy, M.F.; Cosker, K.E.; Machoňová, Olga; Kozmiková, Iryna; Chen, CH.; Pastorino, L.; Asara, J.M.; Cole, A.; Sutherland, C.; Segal, R. A.; Lu, K.P.


    Roč. 13, č. 4 (2015), s. 812-828 ISSN 2211-1247 R&D Projects: GA MŠk(CZ) LK11213; GA MŠk LK21307; GA ČR GA15-03796S; GA MŠk LO1419 Institutional support: RVO:68378050 Keywords : Pin1 * axon guidance * Semaphorin 3A Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.870, year: 2015

  3. Axon tension regulates fasciculation/defasciculation through the control of axon shaft zippering

    Czech Academy of Sciences Publication Activity Database

    Šmít, Daniel; Fouquet, C.; Pincet, F.; Zápotocký, Martin; Trembleau, A.


    Roč. 6, Apr 19 (2017), č. článku e19907. ISSN 2050-084X R&D Projects: GA ČR(CZ) GA14-16755S; GA MŠk(CZ) 7AMB12FR002 Institutional support: RVO:67985823 Keywords : biophysics * cell adhesion * coarsening * developmental biology * mathematical model * mechanical tension * axon guidance Subject RIV: BO - Biophysics OBOR OECD: Biophysics Impact factor: 7.725, year: 2016

  4. Neuron Morphology Influences Axon Initial Segment Plasticity. (United States)

    Gulledge, Allan T; Bravo, Jaime J


    In most vertebrate neurons, action potentials are initiated in the axon initial segment (AIS), a specialized region of the axon containing a high density of voltage-gated sodium and potassium channels. It has recently been proposed that neurons use plasticity of AIS length and/or location to regulate their intrinsic excitability. Here we quantify the impact of neuron morphology on AIS plasticity using computational models of simplified and realistic somatodendritic morphologies. In small neurons (e.g., dentate granule neurons), excitability was highest when the AIS was of intermediate length and located adjacent to the soma. Conversely, neurons having larger dendritic trees (e.g., pyramidal neurons) were most excitable when the AIS was longer and/or located away from the soma. For any given somatodendritic morphology, increasing dendritic membrane capacitance and/or conductance favored a longer and more distally located AIS. Overall, changes to AIS length, with corresponding changes in total sodium conductance, were far more effective in regulating neuron excitability than were changes in AIS location, while dendritic capacitance had a larger impact on AIS performance than did dendritic conductance. The somatodendritic influence on AIS performance reflects modest soma-to-AIS voltage attenuation combined with neuron size-dependent changes in AIS input resistance, effective membrane time constant, and isolation from somatodendritic capacitance. We conclude that the impact of AIS plasticity on neuron excitability will depend largely on somatodendritic morphology, and that, in some neurons, a shorter or more distally located AIS may promote, rather than limit, action potential generation.

  5. Cellular regulation by protein phosphorylation. (United States)

    Fischer, Edmond H


    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert. Copyright © 2012. Published by Elsevier Inc.

  6. Morphology and distribution of chandelier cell axon terminals in the mouse cerebral cortex and claustroamygdaloid complex. (United States)

    Inda, M C; DeFelipe, J; Muñoz, A


    Chandelier cells represent a unique type of cortical gamma-aminobutityric acidergic interneuron whose axon terminals (Ch-terminals) only form synapses with the axon initial segments of some pyramidal cells. Here, we have used immunocytochemistry for the high-affinity plasma membrane transporter GAT-1 and the calcium-binding protein parvalbumin to analyze the morphology and distribution of Ch-terminals in the mouse cerebral cortex and claustroamygdaloid complex. In general, 2 types of Ch-terminals were distinguished on the basis of their size and the density of the axonal boutons that made up the terminal. Simple Ch-terminals were made up of 1 or 2 rows of labeled boutons, each row consisting of only 3-5 boutons. In contrast, complex Ch-terminals were tight cylinder-like structures made up of multiple rows of boutons. Simple Ch-terminals were detected throughout the cerebral cortex and claustroamygdaloid complex, the complex type was only occasionally found in certain regions, whereas in others they were very abundant. These results indicate that there are substantial differences in the morphology and distribution of Ch-terminals between different areas and layers of the mouse cerebral cortex. Furthermore, we suggest that the distribution of complex Ch-terminals may be related to the developmental origin of the different brain regions analyzed.

  7. Axonal Regulation of Central Nervous System Myelination: Structure and Function. (United States)

    Klingseisen, Anna; Lyons, David A


    Approximately half of the human brain consists of myelinated axons. Central nervous system (CNS) myelin is made by oligodendrocytes and is essential for nervous system formation, health, and function. Once thought simply as a static insulator that facilitated rapid impulse conduction, myelin is now known to be made and remodeled in to adult life. Oligodendrocytes have a remarkable capacity to differentiate by default, but many aspects of their development can be influenced by axons. However, how axons and oligodendrocytes interact and cooperate to regulate myelination in the CNS remains unclear. Here, we review recent advances in our understanding of how such interactions generate the complexity of myelination known to exist in vivo. We highlight intriguing results that indicate that the cross-sectional size of an axon alone may regulate myelination to a surprising degree. We also review new studies, which have highlighted diversity in the myelination of axons of different neuronal subtypes and circuits, and structure-function relationships, which suggest that myelinated axons can be exquisitely fine-tuned to mediate precise conduction needs. We also discuss recent advances in our understanding of how neuronal activity regulates CNS myelination, and aim to provide an integrated overview of how axon-oligodendrocyte interactions sculpt neuronal circuit structure and function.

  8. Regeneration of axons in the mouse retina after injury. (United States)

    McConnell, P; Berry, M


    It is generally accepted that most axons in the mammalian CNS show only transient growth in response to injury, and numerous hypotheses have been advanced to account for this phenomenon. Detailed knowledge of the time-course and extent of this so-called 'abortive regeneration' is, however, surprisingly lacking. The retina of the adult albino mouse provides a convenient system in which to quantify the response of central axons to injury, since the retina can be prepared as a whole mount, allowing silver-impregnated axons to be followed along their entire course. Using this experimental model, sprouting of injured axons was observed as early as 14 h post lesion (hpl) with rapid growth (20 micrometers/day on average) continuing until 10 dpl. Thereafter, a decline in the overall growth rate was observed, presumably regenerated sprouts began to degenerate. However, not all axons showed this abortive response: numerous unfasciculated axons continued in random growth until at least 100 dpl. One possible interpretation of these results is that the concept of abortive regeneration of injured axons is untenable in regions of the CNS which are lacking in myelin.

  9. Axonal and presynaptic RNAs are locally transcribed in glial cells. (United States)

    Giuditta, Antonio; Chun, Jong Tai; Eyman, Maria; Cefaliello, Carolina; Bruno, Anna Paola; Crispino, Marianna


    In the last few years, the long-standing opinion that axonal and presynaptic proteins are exclusively derived from the neuron cell body has been substantially modified by the demonstration that active systems of protein synthesis are present in axons and nerve terminals. These observations have raised the issue of the cellular origin of the involved RNAs, which has been generally attributed to the neuron soma. However, data gathered in a number of model systems indicated that axonal RNAs are synthesized in the surrounding glial cells. More recent experiments on the perfused squid giant axon have definitively proved that axoplasmic RNAs are transcribed in periaxonal glia. Their delivery to the axon occurs by a modulatory mechanism based on the release of neurotransmitters from the stimulated axon and on their binding to glial receptors. In additional experiments on squid optic lobe synaptosomes, presynaptic RNA has been also shown to be synthesized locally, presumably in nearby glia. Together with a wealth of literature data, these observations indicate that axons and nerve terminals are endowed with a local system of gene expression that supports the maintenance and plasticity of these neuronal domains.

  10. Propagation of action potentials in inhomogeneous axon regions. (United States)

    Ramón, F; Joyner, R W; Moore, J W


    Described are studies of propagation of action potentials through inhomogenous axon regions through experiments performed on squid giant axons and by computer simulations. The initial speed of propagation of the action potential is dependent upon the stimulus waveform. For a rectangular pulse of current, the action potential travel initally at a high speed that declines over the distance, reaching a constant speed of propagation at about 1-5 resting length constants; this distance depends on the stimulus strength. additional experiments studied the effects of changing the axon diameter and of introducing a temperature step. It was found that the propagated action potential suffers profound modification in shape and velocity as it reaches the region of transition. In both cases, it was possible to obtain reflected action potentials. A region of increased effective diameter was produced experimentally in the squid giant axon by insertion of an axial wire as usually employed in voltage clamps. It was found that the action potential, at the axial wire tip region, undergoes shape changes similar to those obtained tn simulations of a region of increased diameter as in a junction with the axon and soma in motor neurons. It is conducluded that the gaint axon can be used to reproduce simple electrical behaviors in other structures.-Ramón, F., R. W. Joyner and J.W. Moore. Propagation of action potentials in inhomogeneous axon regions.

  11. Axonal Membranes and Their Domains: Assembly and Function of the Axon Initial Segment and Node of Ranvier

    Directory of Open Access Journals (Sweden)

    Andrew D. Nelson


    Full Text Available Neurons are highly specialized cells of the nervous system that receive, process and transmit electrical signals critical for normal brain function. Here, we review the intricate organization of axonal membrane domains that facilitate rapid action potential conduction underlying communication between complex neuronal circuits. Two critical excitable domains of vertebrate axons are the axon initial segment (AIS and the nodes of Ranvier, which are characterized by the high concentrations of voltage-gated ion channels, cell adhesion molecules and specialized cytoskeletal networks. The AIS is located at the proximal region of the axon and serves as the site of action potential initiation, while nodes of Ranvier, gaps between adjacent myelin sheaths, allow rapid propagation of the action potential through saltatory conduction. The AIS and nodes of Ranvier are assembled by ankyrins, spectrins and their associated binding partners through the clustering of membrane proteins and connection to the underlying cytoskeleton network. Although the AIS and nodes of Ranvier share similar protein composition, their mechanisms of assembly are strikingly different. Here we will cover the mechanisms of formation and maintenance of these axonal excitable membrane domains, specifically highlighting the similarities and differences between them. We will also discuss recent advances in super resolution fluorescence imaging which have elucidated the arrangement of the submembranous axonal cytoskeleton revealing a surprising structural organization necessary to maintain axonal organization and function. Finally, human mutations in axonal domain components have been associated with a growing number of neurological disorders including severe cognitive dysfunction, epilepsy, autism, neurodegenerative diseases and psychiatric disorders. Overall, this review highlights the assembly, maintenance and function of axonal excitable domains, particularly the AIS and nodes of

  12. Axon diameter mapping in crossing fibers with diffusion MRI

    DEFF Research Database (Denmark)

    Zhang, Hui; Dyrby, Tim B; Alexander, Daniel C


    tissue than measures derived from diffusion tensor imaging. Most existing techniques for axon diameter mapping assume a single axon orientation in the tissue model, which limits their application to only the most coherently oriented brain white matter, such as the corpus callosum, where the single......This paper proposes a technique for a previously unaddressed problem, namely, mapping axon diameter in crossing fiber regions, using diffusion MRI. Direct measurement of tissue microstructure of this kind using diffusion MRI offers a new class of biomarkers that give more specific information about...... orientation assumption is a reasonable one. However, fiber crossings and other complex configurations are widespread in the brain. In such areas, the existing techniques will fail to provide useful axon diameter indices for any of the individual fiber populations. We propose a novel crossing fiber tissue...

  13. Fiber Optic Detection of Action Potentials in Axons

    National Research Council Canada - National Science Library

    Smela, Elisabeth


    In prior exploratory research, we had designed a fiber optic sensor utilizing a long period Bragg grating for the purpose of detecting action potentials in axons optically, through a change in index...

  14. Spontaneous axonal regeneration in rodent spinal cord after ischemic injury

    DEFF Research Database (Denmark)

    von Euler, Mia; Janson, A M; Larsen, Jytte Overgaard


    cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord......Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total...... length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann...

  15. The nigrostriatal pathway: axonal collateralization and compartmental specificity. (United States)

    Prensa, L; Giménez-Amaya, J M; Parent, A; Bernácer, J; Cebrián, C


    This paper reviews two of the major features of the nigrostriatal pathway, its axonal collateralization, and compartmental specificity, as revealed by single-axon labeling experiments in rodents and immunocytological analysis of human postmortem tissue. The dorsal and ventral tiers of the substantia nigra pars compacta harbor various types of neurons the axons of which branch not only within the striatum but also in other major components of the basal ganglia. Furthermore, some nigrostriatal axons send collaterals both to thalamus and to brainstem pedunculopontine tegmental nucleus. In humans, the compartmental specificity of the nigrostriatal pathway is revealed by the fact that the matrix compartment is densely innervated by dopaminergic fibers, whereas the striosomes display different densities of dopaminergic terminals depending on their location within the striatum. The nigral neurons most severely affected in Parkinson's disease are the ventral tier cells that project to the matrix and form deep clusters in the substantia nigra pars reticulata.

  16. Syndecan Promotes Axon Regeneration by Stabilizing Growth Cone Migration

    Directory of Open Access Journals (Sweden)

    Tyson J. Edwards


    Full Text Available Growth cones facilitate the repair of nervous system damage by providing the driving force for axon regeneration. Using single-neuron laser axotomy and in vivo time-lapse imaging, we show that syndecan, a heparan sulfate (HS proteoglycan, is required for growth cone function during axon regeneration in C. elegans. In the absence of syndecan, regenerating growth cones form but are unstable and collapse, decreasing the effective growth rate and impeding regrowth to target cells. We provide evidence that syndecan has two distinct functions during axon regeneration: (1 a canonical function in axon guidance that requires expression outside the nervous system and depends on HS chains and (2 an intrinsic function in growth cone stabilization that is mediated by the syndecan core protein, independently of HS. Thus, syndecan is a regulator of a critical choke point in nervous system repair.

  17. Fcγ receptor-mediated inflammation inhibits axon regeneration.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

    Full Text Available Anti-glycan/ganglioside antibodies are the most common immune effectors found in patients with Guillain-Barré Syndrome, which is a peripheral autoimmune neuropathy. We previously reported that disease-relevant anti-glycan autoantibodies inhibited axon regeneration, which echo the clinical association of these antibodies and poor recovery in Guillain-Barré Syndrome. However, the specific molecular and cellular elements involved in this antibody-mediated inhibition of axon regeneration are not previously defined. This study examined the role of Fcγ receptors and macrophages in the antibody-mediated inhibition of axon regeneration. A well characterized antibody passive transfer sciatic nerve crush and transplant models were used to study the anti-ganglioside antibody-mediated inhibition of axon regeneration in wild type and various mutant and transgenic mice with altered expression of specific Fcγ receptors and macrophage/microglia populations. Outcome measures included behavior, electrophysiology, morphometry, immunocytochemistry, quantitative real-time PCR, and western blotting. We demonstrate that the presence of autoantibodies, directed against neuronal/axonal cell surface gangliosides, in the injured mammalian peripheral nerves switch the proregenerative inflammatory environment to growth inhibitory milieu by engaging specific activating Fcγ receptors on recruited monocyte-derived macrophages to cause severe inhibition of axon regeneration. Our data demonstrate that the antibody orchestrated Fcγ receptor-mediated switch in inflammation is one mechanism underlying inhibition of axon regeneration. These findings have clinical implications for nerve repair and recovery in antibody-mediated immune neuropathies. Our results add to the complexity of axon regeneration in injured peripheral and central nervous systems as adverse effects of B cells and autoantibodies on neural injury and repair are increasingly recognized.

  18. Modality-Specific Axonal Regeneration: Towards selective regenerative neural interfaces

    Directory of Open Access Journals (Sweden)

    Parisa eLotfi


    Full Text Available Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control of robotic prosthetic devices. However, sensory and motor axons at the neural interface are of mixed submodality types, which difficult the specific recording from motor axons and the eliciting of precise sensory modalities through selective stimulation. Here we evaluated the possibility of using type-specific neurotrophins to preferentially entice the regeneration of defined axonal populations from transected peripheral nerves into separate compartments. Segregation of mixed sensory fibers from dorsal root ganglion neurons was evaluated in vitro by compartmentalized diffusion delivery of nerve growth factor (NGF and neurotrophin-3 (NT-3, to preferentially entice the growth of TrkA+ nociceptive and TrkC+ proprioceptive subsets of sensory neurons, respectively. The average axon length in the NGF channel increased 2.5 fold compared to that in saline or NT-3, whereas the number of branches increased 3 fold in the NT-3 channels. These results were confirmed using a 3-D Y-shaped in vitro assay showing that the arm containing NGF was able to entice a 5-fold increase in axonal length of unbranched fibers. To address if such segregation can be enticed in vivo, a Y-shaped tubing was used to allow regeneration of the transected adult rat sciatic nerve into separate compartments filled with either NFG or NT-3. A significant increase in the number of CGRP+ pain fibers were attracted towards the sural nerve, while N-52+ large diameter axons were observed in the tibial and NT-3 compartments. This study demonstrates the guided enrichment of sensory axons in specific regenerative chambers, and supports the notion that neurotrophic factors can be used to segregate sensory and perhaps motor axons in separate peripheral interfaces.

  19. Modality-specific axonal regeneration: toward selective regenerative neural interfaces. (United States)

    Lotfi, Parisa; Garde, Kshitija; Chouhan, Amit K; Bengali, Ebrahim; Romero-Ortega, Mario I


    Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control of robotic prosthetic devices. However, sensory and motor axons at the neural interface are of mixed sub-modality types, which difficult the specific recording from motor axons and the eliciting of precise sensory modalities through selective stimulation. Here we evaluated the possibility of using type specific neurotrophins to preferentially entice the regeneration of defined axonal populations from transected peripheral nerves into separate compartments. Segregation of mixed sensory fibers from dorsal root ganglion neurons was evaluated in vitro by compartmentalized diffusion delivery of nerve growth factor (NGF) and neurotrophin-3 (NT-3), to preferentially entice the growth of TrkA+ nociceptive and TrkC+ proprioceptive subsets of sensory neurons, respectively. The average axon length in the NGF channel increased 2.5-fold compared to that in saline or NT-3, whereas the number of branches increased threefold in the NT-3 channels. These results were confirmed using a 3D "Y"-shaped in vitro assay showing that the arm containing NGF was able to entice a fivefold increase in axonal length of unbranched fibers. To address if such segregation can be enticed in vivo, a "Y"-shaped tubing was used to allow regeneration of the transected adult rat sciatic nerve into separate compartments filled with either NFG or NT-3. A significant increase in the number of CGRP+ pain fibers were attracted toward the sural nerve, while N-52+ large-diameter axons were observed in the tibial and NT-3 compartments. This study demonstrates the guided enrichment of sensory axons in specific regenerative chambers, and supports the notion that neurotrophic factors can be used to segregate sensory and perhaps motor axons in separate peripheral interfaces.

  20. MR imaging of a diffuse axonal injury

    International Nuclear Information System (INIS)

    Inoue, Yukiya; Okamoto, Hisayo; Mitsushima, Minoru; Hori, Tomokatsu; Sasaki, Mamoru; Teraoka, Akira.


    Six patients who had been diagnosed as having so-called a 'Diffuse Axonal Injury (DAI)' were examined by means of Magnetic Resonance Imaging (Yokogawa Resona 0.5T and Shimadzu SMT 50A). MRI revealed clear evidence of injured white matter in these patients, while X-ray CT scanning could not demonstrate such lesions definitely. The patients consisted of three adults and three adolescents. They had been injured by traffic accidents or falls. Every patient had lost consciousness immediately, and their coma had continued for at least two weeks after the trauma. X-ray CT scanning demonstrated no complicated lesion, such as intracranial hematoma or brain edema, resulting in increased intracranial pressure and cerebral herniation. In all of the patients, injuries of the deep white matter (corpus callosum, upper pons, or internal capsule, for example) were clearly found by T 2 -weighted imaging. Because these lesions had characteristic features in their localation, as has been described by Adams et al. these patients were diagnosed as having DAI. Also, it was interesting that the focal neurological deficits of these patients correlated well with the local injuries of the white matter. The three young patients recovered to various degrees, but the three adults passed into a vegetative state. The prognosis of the patients seemed to be determined by their age. Because the clinical diagnosis of DAI is controversial, the use of MRI will help in its clinical diagnosis and analysis. (author)

  1. MR imaging of a diffuse axonal injury

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Yukiya; Okamoto, Hisayo; Mitsushima, Minoru; Hori, Tomokatsu (Tottori Univ., Yonago (Japan). School of Medicine); Sasaki, Mamoru; Teraoka, Akira


    Six patients who had been diagnosed as having so-called a 'Diffuse Axonal Injury (DAI)' were examined by means of Magnetic Resonance Imaging (Yokogawa Resona 0.5T and Shimadzu SMT 50A). MRI revealed clear evidence of injured white matter in these patients, while X-ray CT scanning could not demonstrate such lesions definitely. The patients consisted of three adults and three adolescents. They had been injured by traffic accidents or falls. Every patient had lost consciousness immediately, and their coma had continued for at least two weeks after the trauma. X-ray CT scanning demonstrated no complicated lesion, such as intracranial hematoma or brain edema, resulting in increased intracranial pressure and cerebral herniation. In all of the patients, injuries of the deep white matter (corpus callosum, upper pons, or internal capsule, for example) were clearly found by T{sub 2}-weighted imaging. Because these lesions had characteristic features in their localation, as has been described by Adams et al. these patients were diagnosed as having DAI. Also, it was interesting that the focal neurological deficits of these patients correlated well with the local injuries of the white matter. The three young patients recovered to various degrees, but the three adults passed into a vegetative state. The prognosis of the patients seemed to be determined by their age. Because the clinical diagnosis of DAI is controversial, the use of MRI will help in its clinical diagnosis and analysis. (author).

  2. c-Abl, Lamellipodin, and Ena/VASP proteins cooperate in dorsal ruffling of fibroblasts and axonal morphogenesis. (United States)

    Michael, Magdalene; Vehlow, Anne; Navarro, Christel; Krause, Matthias


    Tight regulation of cell motility is essential for many physiological processes, such as formation of a functional nervous system and wound healing. Drosophila Abl negatively regulates the actin cytoskeleton effector protein Ena during neuronal development in flies, and it has been postulated that this may occur through an unknown intermediary. Lamellipodin (Lpd) regulates cell motility and recruits Ena/VASP proteins (Ena, Mena, VASP, EVL) to the leading edge of cells. However, the regulation of this recruitment has remained unsolved. Here we show that Lpd is a substrate of Abl kinases and binds to the Abl SH2 domain. Phosphorylation of Lpd positively regulates the interaction between Lpd and Ena/VASP proteins. Consistently, efficient recruitment of Mena and EVL to Lpd at the leading edge requires Abl kinases. Furthermore, transient Lpd phosphorylation by Abl kinases upon netrin-1 stimulation of primary cortical neurons positively correlates with an increase in Lpd-Mena coprecipitation. Lpd is also transiently phosphorylated by Abl kinases upon platelet-derived growth factor (PDGF) stimulation, regulates PDGF-induced dorsal ruffling of fibroblasts and axonal morphogenesis, and cooperates with c-Abl in an Ena/VASP-dependent manner. Our findings suggest that Abl kinases positively regulate Lpd-Ena/VASP interaction, Ena/VASP recruitment to Lpd at the leading edge, and Lpd-Ena/VASP function in axonal morphogenesis and in PDGF-induced dorsal ruffling. Our data do not support the suggested negative regulatory role of Abl for Ena. Instead, we propose that Lpd is the hitherto unknown intermediary between Abl and Ena/VASP proteins. (c) 2010 Elsevier Ltd. All rights reserved.

  3. Developmental time windows for axon growth influence neuronal network topology. (United States)

    Lim, Sol; Kaiser, Marcus


    Early brain connectivity development consists of multiple stages: birth of neurons, their migration and the subsequent growth of axons and dendrites. Each stage occurs within a certain period of time depending on types of neurons and cortical layers. Forming synapses between neurons either by growing axons starting at similar times for all neurons (much-overlapped time windows) or at different time points (less-overlapped) may affect the topological and spatial properties of neuronal networks. Here, we explore the extreme cases of axon formation during early development, either starting at the same time for all neurons (parallel, i.e., maximally overlapped time windows) or occurring for each neuron separately one neuron after another (serial, i.e., no overlaps in time windows). For both cases, the number of potential and established synapses remained comparable. Topological and spatial properties, however, differed: Neurons that started axon growth early on in serial growth achieved higher out-degrees, higher local efficiency and longer axon lengths while neurons demonstrated more homogeneous connectivity patterns for parallel growth. Second, connection probability decreased more rapidly with distance between neurons for parallel growth than for serial growth. Third, bidirectional connections were more numerous for parallel growth. Finally, we tested our predictions with C. elegans data. Together, this indicates that time windows for axon growth influence the topological and spatial properties of neuronal networks opening up the possibility to a posteriori estimate developmental mechanisms based on network properties of a developed network.

  4. Parametric Probability Distribution Functions for Axon Diameters of Corpus Callosum

    Directory of Open Access Journals (Sweden)

    Farshid eSepehrband


    Full Text Available Axon diameter is an important neuroanatomical characteristic of the nervous system that alters in the course of neurological disorders such as multiple sclerosis. Axon diameters vary, even within a fiber bundle, and are not normally distributed. An accurate distribution function is therefore beneficial, either to describe axon diameters that are obtained from a direct measurement technique (e.g., microscopy, or to infer them indirectly (e.g., using diffusion-weighted MRI. The gamma distribution is a common choice for this purpose (particularly for the inferential approach because it resembles the distribution profile of measured axon diameters which has been consistently shown to be non-negative and right-skewed. In this study we compared a wide range of parametric probability distribution functions against empirical data obtained from electron microscopy images. We observed that the gamma distribution fails to accurately describe the main characteristics of the axon diameter distribution, such as location and scale of the mode and the profile of distribution tails. We also found that the generalized extreme value distribution consistently fitted the measured distribution better than other distribution functions. This suggests that there may be distinct subpopulations of axons in the corpus callosum, each with their own distribution profiles. In addition, we observed that several other distributions outperformed the gamma distribution, yet had the same number of unknown parameters; these were the inverse Gaussian, log normal, log logistic and Birnbaum-Saunders distributions.

  5. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    International Nuclear Information System (INIS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir


    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

  6. Tyrosine phosphorylation in signal transduction

    International Nuclear Information System (INIS)

    Roberts, T.M.; Kaplan, D.; Morgan, W.; Keller, T.; Mamon, H.; Piwnica-Worms, H.; Druker, B.; Whitman, M.; Morrison, D.; Cohen, B.; Schaffhausen, B.; Cantley, L.; Rapp, U.


    Recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation the authors have employed the following strategy. First, they have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60 c-src , the CSF- receptor, or the platelet-derived growth factor (PDGF) receptor. Second, they have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues. In this endeavor, they are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the genes or cDNAs for the substrates and overproduce the relevant proteins for studies in vitro in defined systems. This paper describes the first and second aspects of this strategy, the identification and characterization of novel substrate molecules

  7. Axonal accumulation of synaptic markers in APP transgenic Drosophila depends on the NPTY motif and is paralleled by defects in synaptic plasticity

    DEFF Research Database (Denmark)

    Rusu, Patricia; Jansen, Anna; Soba, Peter


    . Specifically, axonal transport defects have been reported in AD animal models, including mice and flies that overexpress APP and tau. Here we demonstrate that the APP-induced traffic jam of vesicles in peripheral nerves of Drosophila melanogaster larvae depends on the four residues NPTY motif in the APP...

  8. Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide

    International Nuclear Information System (INIS)

    Hayes, G.R.; Lockwood, D.H.


    The oxidant H 2 O 2 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H 2 O 2 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the β subunit of the insulin receptor was increased. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport. Phosphoamino acid analysis of insulin receptors from H 2 O 2 -treated adipocytes showed that 32 P incorporation into phosphotyrosine and phosphoserine residues of the β subunit was enhanced. Furthermore, partially purified receptors from H 2 O 2 -treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu 80 Tyr 20 . To define the factors involved in H 2 O 2 's effect, the authors have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H 2 O 2 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H 2 O 2 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H 2 O 2 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H 2 O 2 on the insulin receptor and requires nonplasma membrane cellular constituents

  9. Sensory axon-derived neuregulin-1 is required for axoglial signaling and normal sensory function but not for long-term axon maintenance

    DEFF Research Database (Denmark)

    Fricker, F.R.; Zhu, N.; Tsantoulas, C.


    to represent large-diameter axons that have failed to myelinate. Conditional neuregulin-1 ablation resulted in a reduced sensitivity to noxious mechanical stimuli. These findings emphasize the importance of neuregulin-1 in mediating the signaling between axons and both myelinating and nonmyelinating Schwann...... cells required for normal sensory function. Sensory neuronal survival and axonal maintenance, however, are not dependent on axon-derived neuregulin-1 signaling in adulthood Udgivelsesdato: 2009/6/17...

  10. Binding of the Substrate Analogue Perseitol to Phosphorylated and Unphosphorylated Enzyme IImtl of the Phosphoenolpyruvate-Dependent Phosphotransferase System of Escherichia coli

    NARCIS (Netherlands)

    Lolkema, Juke S.; Wartna, Ellen S.; Robillard, George T.


    Enzyme IImtl catalyzes the concomitant transport and phosphorylation of the hexitol mannitol. Here we demonstrate that the heptitol perseitol is not phosphorylated and not transported by the enzyme. However, the enzyme binds perseitol with an affinity comparable to the affinity for mannitol.

  11. Modelling in vivo action potential propagation along a giant axon. (United States)

    George, Stuart; Foster, Jamie M; Richardson, Giles


    A partial differential equation model for the three-dimensional current flow in an excitable, unmyelinated axon is considered. Where the axon radius is significantly below a critical value R(crit) (that depends upon intra- and extra-cellular conductivity and ion channel conductance) the resistance of the intracellular space is significantly higher than that of the extracellular space, such that the potential outside the axon is uniformly small whilst the intracellular potential is approximated by the transmembrane potential. In turn, since the current flow is predominantly axial, it can be shown that the transmembrane potential is approximated by a solution to the one-dimensional cable equation. It is noted that the radius of the squid giant axon, investigated by (Hodgkin and Huxley 1952e), lies close to R(crit). This motivates us to apply the three-dimensional model to the squid giant axon and compare the results thus found to those obtained using the cable equation. In the context of the in vitro experiments conducted in (Hodgkin and Huxley 1952e) we find only a small difference between the wave profiles determined using these two different approaches and little difference between the speeds of action potential propagation predicted. This suggests that the cable equation approximation is accurate in this scenario. However when applied to the it in vivo setting, in which the conductivity of the surrounding tissue is considerably lower than that of the axoplasm, there are marked differences in both wave profile and speed of action potential propagation calculated using the two approaches. In particular, the cable equation significantly over predicts the increase in the velocity of propagation as axon radius increases. The consequences of these results are discussed in terms of the evolutionary costs associated with increasing the speed of action potential propagation by increasing axon radius.

  12. Molecular Disorganization of Axons Adjacent to Human Cortical Microinfarcts

    Directory of Open Access Journals (Sweden)

    Hamza Coban


    Full Text Available Cortical microinfarcts (CMIs are microscopically identified wedge-shaped ischemic lesions that occur at or near the cortical surface and result from occlusion of penetrating arterioles. These microscopic lesions can be observed with high-resolution magnetic resonance imaging in aging brains and in patients with cerebrovascular disease. Recent studies have suggested that strategically located microinfarcts strongly correlate with cognitive deficits, which can contribute to Alzheimer’s disease as well as other forms of dementia. We have recently shown that the molecular organization of axons into functional microdomains is altered in areas adjacent to white matter lacunar and microinfarcts, creating a peri-infarct penumbral injury in surviving axons. Whether similar changes in nodal, adjacent paranodal, and proximal axon initial segment molecular organization occur in the cortex adjacent to human CMIs is not known. Paraffin-embedded sections of autopsy brain tissue from five patients with CMIs were immunofluorescently labeled for nodal and paranodal markers including beta-IV spectrin, ankyrin-G, and contactin-associated protein. High magnification images from the peri-infarct cortical tissue were generated using confocal microscopy. In surviving cortical tissue adjacent to microinfarcts, we observed a dramatic loss of axon initial segments, suggesting that neuronal firing capacity in adjacent cortical tissue is likely compromised. The number of identifiable nodal/paranodal complexes in surviving cortical tissue is reduced adjacent to microinfarcts, while the average paranodal length is increased indicating a breakdown of axoglial contact. This axonal microdomain disorganization occurs in the relative absence of changes in the structural integrity of myelinated axons as measured by myelin basic protein and neurofilament staining. These findings indicate that the molecular organization of surviving axons adjacent to human CMIs is abnormal

  13. Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a. (United States)

    Wang, Tong; Martin, Sally; Papadopulos, Andreas; Harper, Callista B; Mavlyutov, Timur A; Niranjan, Dhevahi; Glass, Nick R; Cooper-White, Justin J; Sibarita, Jean-Baptiste; Choquet, Daniel; Davletov, Bazbek; Meunier, Frédéric A


    Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity. Copyright © 2015 the authors 0270-6474/15/356179-16$15.00/0.

  14. Protein phosphorylations in poliovirus infected cells. (United States)

    James, L A; Tershak, D R


    In vivo phosphorylation of proteins that are associated with polysomes of poliovirus-infected VERO (African green monkey kidney) and HeLa (Henrietta Lacks) cells differed from phosphorylations observed with uninfected cells that were fed fresh medium. With both types of cells infection stimulated phosphorylation of proteins with molecular weights of 40 000-41 000, 39 000, 34 000, 32 000, and 24 000. Similarities of phosphorylations in VERO and HeLa cells suggest that they are a specific consequence of infection and might serve a regulatory function during protein synthesis.

  15. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.


    of protein-tyrosine phosphorylation. We discuss the approaches currently used to chart this network: ranging from studies of substrate specifi city and the physiological role of tyrosine phosphorylation of individual enzymes to the global approaches at the level of systems biology....... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

  16. Myelinated sensory and alpha motor axon regeneration in peripheral nerve neuromas (United States)

    Macias, M. Y.; Lehman, C. T.; Sanger, J. R.; Riley, D. A.


    Histochemical staining for carbonic anhydrase and cholinesterase (CE) activities was used to analyze sensory and motor axon regeneration, respectively, during neuroma formation in transected and tube-encapsulated peripheral nerves. Median-ulnar and sciatic nerves in the rodent model permitted testing whether a 4 cm greater distance of the motor neuron soma from axotomy site or intrinsic differences between motor and sensory neurons influenced regeneration and neuroma formation 10, 30, and 90 days later. Ventral root radiculotomy confirmed that CE-stained axons were 97% alpha motor axons. Distance significantly delayed axon regeneration. When distance was negligible, sensory axons grew out sooner than motor axons, but motor axons regenerated to a greater quantity. These results indicate regeneration differences between axon subtypes and suggest more extensive branching of motor axons within the neuroma. Thus, both distance from injury site to soma and inherent motor and sensory differences should be considered in peripheral nerve repair strategies.

  17. Cortical Interneuron Subtypes Vary in Their Axonal Action Potential Properties. (United States)

    Casale, Amanda E; Foust, Amanda J; Bal, Thierry; McCormick, David A


    The role of interneurons in cortical microcircuits is strongly influenced by their passive and active electrical properties. Although different types of interneurons exhibit unique electrophysiological properties recorded at the soma, it is not yet clear whether these differences are also manifested in other neuronal compartments. To address this question, we have used voltage-sensitive dye to image the propagation of action potentials into the fine collaterals of axons and dendrites in two of the largest cortical interneuron subtypes in the mouse: fast-spiking interneurons, which are typically basket or chandelier neurons; and somatostatin containing interneurons, which are typically regular spiking Martinotti cells. We found that fast-spiking and somatostatin-expressing interneurons differed in their electrophysiological characteristics along their entire dendrosomatoaxonal extent. The action potentials generated in the somata and axons, including axon collaterals, of somatostatin-expressing interneurons are significantly broader than those generated in the same compartments of fast-spiking inhibitory interneurons. In addition, action potentials back-propagated into the dendrites of somatostatin-expressing interneurons much more readily than fast-spiking interneurons. Pharmacological investigations suggested that axonal action potential repolarization in both cell types depends critically upon Kv1 channels, whereas the axonal and somatic action potentials of somatostatin-expressing interneurons also depend on BK Ca(2+)-activated K(+) channels. These results indicate that the two broad classes of interneurons studied here have expressly different subcellular physiological properties, allowing them to perform unique computational roles in cortical circuit operations. Neurons in the cerebral cortex are of two major types: excitatory and inhibitory. The proper balance of excitation and inhibition in the brain is critical for its operation. Neurons contain three main

  18. SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J


    spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

  19. Overcoming Monocarboxylate Transporter 8 (MCT8-Deficiency to Promote Human Oligodendrocyte Differentiation and Myelination

    Directory of Open Access Journals (Sweden)

    Jae Young Lee


    Full Text Available Cell membrane thyroid hormone (TH transport can be facilitated by the monocarboxylate transporter 8 (MCT8, encoded by the solute carrier family 16 member 2 (SLC16A2 gene. Human mutations of the gene, SLC16A2, result in the X-linked-inherited psychomotor retardation and hypomyelination disorder, Allan-Herndon-Dudley syndrome (AHDS. We posited that abrogating MCT8-dependent TH transport limits oligodendrogenesis and myelination. We show that human oligodendrocytes (OL, derived from the NKX2.1-GFP human embryonic stem cell (hESC reporter line, express MCT8. Moreover, treatment of these cultures with DITPA (an MCT8-independent TH analog, up-regulates OL differentiation transcription factors and myelin gene expression. DITPA promotes hESC-derived OL myelination of retinal ganglion axons in co-culture. Pharmacological and genetic blockade of MCT8 induces significant OL apoptosis, impairing myelination. DITPA treatment limits OL apoptosis mediated by SLC16A2 down-regulation primarily signaling through AKT phosphorylation, driving myelination. Our results highlight the potential role of MCT8 in TH transport for human OL development and may implicate DITPA as a promising treatment for developmentally-regulated myelination in AHDS.

  20. Overcoming Monocarboxylate Transporter 8 (MCT8)-Deficiency to Promote Human Oligodendrocyte Differentiation and Myelination. (United States)

    Lee, Jae Young; Kim, Min Joung; Deliyanti, Devy; Azari, Michael F; Rossello, Fernando; Costin, Adam; Ramm, Georg; Stanley, Edouard G; Elefanty, Andrew G; Wilkinson-Berka, Jennifer L; Petratos, Steven


    Cell membrane thyroid hormone (TH) transport can be facilitated by the monocarboxylate transporter 8 (MCT8), encoded by the solute carrier family 16 member 2 (SLC16A2) gene. Human mutations of the gene, SLC16A2, result in the X-linked-inherited psychomotor retardation and hypomyelination disorder, Allan-Herndon-Dudley syndrome (AHDS). We posited that abrogating MCT8-dependent TH transport limits oligodendrogenesis and myelination. We show that human oligodendrocytes (OL), derived from the NKX2.1-GFP human embryonic stem cell (hESC) reporter line, express MCT8. Moreover, treatment of these cultures with DITPA (an MCT8-independent TH analog), up-regulates OL differentiation transcription factors and myelin gene expression. DITPA promotes hESC-derived OL myelination of retinal ganglion axons in co-culture. Pharmacological and genetic blockade of MCT8 induces significant OL apoptosis, impairing myelination. DITPA treatment limits OL apoptosis mediated by SLC16A2 down-regulation primarily signaling through AKT phosphorylation, driving myelination. Our results highlight the potential role of MCT8 in TH transport for human OL development and may implicate DITPA as a promising treatment for developmentally-regulated myelination in AHDS. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  1. Functional complexity of the axonal growth cone: a proteomic analysis.

    Directory of Open Access Journals (Sweden)

    Adriana Estrada-Bernal

    Full Text Available The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ≥ 99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions.

  2. Subtypes of GABAergic neurons project axons in the neocortex

    Directory of Open Access Journals (Sweden)

    Shigeyoshi Higo


    Full Text Available γ-aminobutyric acid (GABAergic neurons in the neocortex have been regarded as interneurons and speculated to modulate the activity of neurons locally. Recently, however, several experiments revealed that neuronal nitric oxide synthase (nNOS-positive GABAergic neurons project cortico-cortically with long axons. In this study, we illustrate Golgi-like images of the nNOS-positive GABAergic neurons using a nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d reaction and follow the emanating axon branches in cat brain sections. These axon branches projected cortico-cortically with other non-labeled arcuate fibers, contra-laterally via the corpus callosum and anterior commissure. The labeled fibers were not limited to the neocortex but found also in the fimbria of the hippocampus. In order to have additional information on these GABAergic neuron projections, we investigated green fluorescent protein (GFP-labeled GABAergic neurons in GAD67-Cre knock-in / GFP Cre-reporter mice. GFP-labeled axons emanate densely, especially in the fimbria, a small number in the anterior commissure, and very sparsely in the corpus callosum. These two different approaches confirm that not only nNOS-positive GABAergic neurons but also other subtypes of GABAergic neurons project long axons in the cerebral cortex and are in a position to be involved in information processing.

  3. Fast and reliable identification of axons, axon initial segments and dendrites with local field potential recording

    DEFF Research Database (Denmark)

    Petersen, Anders V.; Johansen, Emil O.; Perrier, Jean-Francois


    The axon initial segment (AIS) is an essential neuronal compartment. It is usually where action potentials are initiated. Recent studies demonstrated that the AIS is a plastic structure that can be regulated by neuronal activity and by the activation of metabotropic receptors. Studying the AIS...... in live tissue can be difficult because its identification is not always reliable. Here we provide a new technique allowing a fast and reliable identification of the AIS in live brain slice preparations. By simultaneous recording of extracellular local field potentials and whole-cell patch-clamp recording...... of neurons, we can detect sinks caused by inward currents flowing across the membrane. We determine the location of the AIS by comparing the timing of these events with the action potential. We demonstrate that this method allows the unequivocal identification of the AIS of different types of neurons from...

  4. Fast and reliable identification of axons, axon initial segments and dendrites with local field potential recording

    Directory of Open Access Journals (Sweden)

    Anders Victor ePetersen


    Full Text Available The axon initial segment (AIS is an essential neuronal compartment. It is usually where action potentials are initiated. Recent studies demonstrated that the AIS is a plastic structure that can be regulated by neuronal activity and by the activation of metabotropic receptors. Studying the AIS in live tissue can be difficult because its identification is not always reliable. Here we provide a new technique allowing a fast and reliable identification of the AIS in live brain slice preparations. By simultaneous recoding of extracellular local field potentials and whole-cell patch-clamp recording of neurons, we can detect sinks caused by inward currents flowing across the membrane. We determine the location of the AIS by comparing the timing of these events with the action potential. We demonstrate that this method allows the unequivocal identification of the AIS of different types of neurons from the brain.

  5. Mapping protein phosphorylation in zebrafish development

    NARCIS (Netherlands)

    Lemeer, S.M.


    Mapping protein phosphorylation in zebrafish development Reversible protein phosphorylation plays a key role in signaling processes that are vital for a cell and organism. It provides a rapid switch for protein activity as it often changes the conformation and function of a protein in the cell.

  6. Perilesional edema in radiation necrosis reflects axonal degeneration

    International Nuclear Information System (INIS)

    Perez-Torres, Carlos J; Yuan, Liya; Schmidt, Robert E; Rich, Keith M; Ackerman, Joseph JH; Garbow, Joel R


    Recently, we characterized a Gamma Knife® radiation necrosis mouse model with various magnetic resonance imaging (MRI) protocols to identify biomarkers useful in differentiation from tumors. Though the irradiation was focal to one hemisphere, a contralateral injury was observed that appeared to be localized in the white matter only. Interestingly, this injury was identifiable in T2-weighted images, apparent diffusion coefficient (ADC), and magnetization transfer ratio (MTR) maps, but not on post-contrast T1-weighted images. This observation of edema independent of vascular changes is akin to the perilesional edema seen in clinical radiation necrosis. The pathology underlying the observed white-matter MRI changes was explored by performing immunohistochemistry for healthy axons and myelin. The presence of both healthy axons and myelin was reduced in the contralateral white-matter lesion. Based on our immunohistochemical findings, the contralateral white-matter injury is most likely due to axonal degeneration

  7. The nano-architecture of the axonal cytoskeleton. (United States)

    Leterrier, Christophe; Dubey, Pankaj; Roy, Subhojit


    The corporeal beauty of the neuronal cytoskeleton has captured the imagination of generations of scientists. One of the easiest cellular structures to visualize by light microscopy, its existence has been known for well over 100 years, yet we have only recently begun to fully appreciate its intricacy and diversity. Recent studies combining new probes with super-resolution microscopy and live imaging have revealed surprising details about the axonal cytoskeleton and, in particular, have discovered previously unknown actin-based structures. Along with traditional electron microscopy, these newer techniques offer a nanoscale view of the axonal cytoskeleton, which is important for our understanding of neuronal form and function, and lay the foundation for future studies. In this Review, we summarize existing concepts in the field and highlight contemporary discoveries that have fundamentally altered our perception of the axonal cytoskeleton.

  8. The axon-protective WLD(S) protein partially rescues mitochondrial respiration and glycolysis after axonal injury. (United States)

    Godzik, Katharina; Coleman, Michael P


    The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases.

  9. Axoplasmic RNA species synthesized in the isolated squid giant axon. (United States)

    Rapallino, M V; Cupello, A; Giuditta, A


    Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [3H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)+ RNA in all tissue samples. Radioactive poly(A)+ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)+ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.

  10. Pannexin 1 Modulates Axonal Growth in Mouse Peripheral Nerves

    Directory of Open Access Journals (Sweden)

    Steven M. Horton


    Full Text Available The pannexin family of channels consists of three members—pannexin-1 (Panx1, pannexin-2 (Panx2, and pannexin-3 (Panx3 that enable the exchange of metabolites and signaling molecules between intracellular and extracellular compartments. Pannexin-mediated release of intracellular ATP into the extracellular space has been tied to a number of cellular activities, primarily through the activity of type P2 purinergic receptors. Previous work indicates that the opening of Panx1 channels and activation of purinergic receptors by extracellular ATP may cause inflammation and apoptosis. In the CNS (central nervous system and PNS (peripheral nervous system, coupled pannexin, and P2 functions have been linked to peripheral sensitization (pain pathways. Purinergic pathways are also essential for other critical processes in the PNS, including myelination and neurite outgrowth. However, whether such pathways are pannexin-dependent remains to be determined. In this study, we use a Panx1 knockout mouse model and pharmacological inhibitors of the Panx1 and the ATP-mediated signaling pathway to fill gaps in our understanding of Panx1 localization in peripheral nerves, roles for Panx1 in axonal outgrowth and myelination, and neurite extension. Our data show that Panx1 is localized to axonal, myelin, and vascular compartments of the peripheral nerves. Knockout of Panx1 gene significantly increased axonal caliber in vivo and axonal growth rate in cultured dorsal root ganglia (DRG neurons. Furthermore, genetic knockout of Panx1 or inhibition of components of purinergic signaling, by treatment with probenecid and apyrase, resulted in denser axonal outgrowth from cultured DRG explants compared to untreated wild-types. Our findings suggest that Panx1 regulates axonal growth in the peripheral nervous system.

  11. Uncovering sensory axonal dysfunction in asymptomatic type 2 diabetic neuropathy.

    Directory of Open Access Journals (Sweden)

    Jia-Ying Sung

    Full Text Available This study investigated sensory and motor nerve excitability properties to elucidate the development of diabetic neuropathy. A total of 109 type 2 diabetes patients were recruited, and 106 were analyzed. According to neuropathy severity, patients were categorized into G0, G1, and G2+3 groups using the total neuropathy score-reduced (TNSr. Patients in the G0 group were asymptomatic and had a TNSr score of 0. Sensory and motor nerve excitability data from diabetic patients were compared with data from 33 healthy controls. Clinical assessment, nerve conduction studies, and sensory and motor nerve excitability testing data were analyzed to determine axonal dysfunction in diabetic neuropathy. In the G0 group, sensory excitability testing revealed increased stimulus for the 50% sensory nerve action potential (P<0.05, shortened strength-duration time constant (P<0.01, increased superexcitability (P<0.01, decreased subexcitability (P<0.05, decreased accommodation to depolarizing current (P<0.01, and a trend of decreased accommodation to hyperpolarizing current in threshold electrotonus. All the changes progressed into G1 (TNSr 1-8 and G2+3 (TNSr 9-24 groups. In contrast, motor excitability only had significantly increased stimulus for the 50% compound motor nerve action potential (P<0.01 in the G0 group. This study revealed that the development of axonal dysfunction in sensory axons occurred prior to and in a different fashion from motor axons. Additionally, sensory nerve excitability tests can detect axonal dysfunction even in asymptomatic patients. These insights further our understanding of diabetic neuropathy and enable the early detection of sensory axonal abnormalities, which may provide a basis for neuroprotective therapeutic approaches.

  12. Motoneuron axon pathfinding errors in zebrafish: Differential effects related to concentration and timing of nicotine exposure

    International Nuclear Information System (INIS)

    Menelaou, Evdokia; Paul, Latoya T.; Perera, Surangi N.; Svoboda, Kurt R.


    Nicotine exposure during embryonic stages of development can affect many neurodevelopmental processes. In the developing zebrafish, exposure to nicotine was reported to cause axonal pathfinding errors in the later born secondary motoneurons (SMNs). These alterations in SMN axon morphology coincided with muscle degeneration at high nicotine concentrations (15–30 μM). Previous work showed that the paralytic mutant zebrafish known as sofa potato exhibited nicotine-induced effects onto SMN axons at these high concentrations but in the absence of any muscle deficits, indicating that pathfinding errors could occur independent of muscle effects. In this study, we used varying concentrations of nicotine at different developmental windows of exposure to specifically isolate its effects onto subpopulations of motoneuron axons. We found that nicotine exposure can affect SMN axon morphology in a dose-dependent manner. At low concentrations of nicotine, SMN axons exhibited pathfinding errors, in the absence of any nicotine-induced muscle abnormalities. Moreover, the nicotine exposure paradigms used affected the 3 subpopulations of SMN axons differently, but the dorsal projecting SMN axons were primarily affected. We then identified morphologically distinct pathfinding errors that best described the nicotine-induced effects on dorsal projecting SMN axons. To test whether SMN pathfinding was potentially influenced by alterations in the early born primary motoneuron (PMN), we performed dual labeling studies, where both PMN and SMN axons were simultaneously labeled with antibodies. We show that only a subset of the SMN axon pathfinding errors coincided with abnormal PMN axonal targeting in nicotine-exposed zebrafish. We conclude that nicotine exposure can exert differential effects depending on the levels of nicotine and developmental exposure window. - Highlights: • Embryonic nicotine exposure can specifically affect secondary motoneuron axons in a dose-dependent manner.

  13. Motoneuron axon pathfinding errors in zebrafish: Differential effects related to concentration and timing of nicotine exposure

    Energy Technology Data Exchange (ETDEWEB)

    Menelaou, Evdokia; Paul, Latoya T. [Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 (United States); Perera, Surangi N. [Joseph J. Zilber School of Public Health, University of Wisconsin — Milwaukee, Milwaukee, WI 53205 (United States); Svoboda, Kurt R., E-mail: [Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 (United States); Joseph J. Zilber School of Public Health, University of Wisconsin — Milwaukee, Milwaukee, WI 53205 (United States)


    Nicotine exposure during embryonic stages of development can affect many neurodevelopmental processes. In the developing zebrafish, exposure to nicotine was reported to cause axonal pathfinding errors in the later born secondary motoneurons (SMNs). These alterations in SMN axon morphology coincided with muscle degeneration at high nicotine concentrations (15–30 μM). Previous work showed that the paralytic mutant zebrafish known as sofa potato exhibited nicotine-induced effects onto SMN axons at these high concentrations but in the absence of any muscle deficits, indicating that pathfinding errors could occur independent of muscle effects. In this study, we used varying concentrations of nicotine at different developmental windows of exposure to specifically isolate its effects onto subpopulations of motoneuron axons. We found that nicotine exposure can affect SMN axon morphology in a dose-dependent manner. At low concentrations of nicotine, SMN axons exhibited pathfinding errors, in the absence of any nicotine-induced muscle abnormalities. Moreover, the nicotine exposure paradigms used affected the 3 subpopulations of SMN axons differently, but the dorsal projecting SMN axons were primarily affected. We then identified morphologically distinct pathfinding errors that best described the nicotine-induced effects on dorsal projecting SMN axons. To test whether SMN pathfinding was potentially influenced by alterations in the early born primary motoneuron (PMN), we performed dual labeling studies, where both PMN and SMN axons were simultaneously labeled with antibodies. We show that only a subset of the SMN axon pathfinding errors coincided with abnormal PMN axonal targeting in nicotine-exposed zebrafish. We conclude that nicotine exposure can exert differential effects depending on the levels of nicotine and developmental exposure window. - Highlights: • Embryonic nicotine exposure can specifically affect secondary motoneuron axons in a dose-dependent manner.

  14. IFNgamma enhances microglial reactions to hippocampal axonal degeneration

    DEFF Research Database (Denmark)

    Jensen, M B; Hegelund, I V; Lomholt, N D


    Glial reactivity is implicated in CNS repair and regenerative responses. Microglia, the cells responding earliest to axonal injury, produce tumor necrosis factor-alpha (TNFalpha), a cytokine with both cytopathic and neuroprotective effects. We have studied activation of hippocampal microglia...... periods. Message for the immune cytokine interferon-gamma (IFNgamma) was undetectable, and glial reactivity to axonal lesions occurred as normal in IFNgamma-deficient mice. Microglial responses to lesion-induced neuronal injury were markedly enhanced in myelin basic protein promoter-driven transgenic mice...

  15. Axon diameter and intra-axonal volume fraction of the corticospinal tract in idiopathic normal pressure hydrocephalus measured by q-space imaging.

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    Kouhei Kamiya

    Full Text Available PURPOSE: Previous studies suggest that compression and stretching of the corticospinal tract (CST potentially cause treatable gait disturbance in patients with idiopathic normal pressure hydrocephalus (iNPH. Measurement of axon diameter with diffusion MRI has recently been used to investigate microstructural alterations in neurological diseases. In this study, we investigated alterations in the axon diameter and intra-axonal fraction of the CST in iNPH by q-space imaging (QSI analysis. METHODS: Nineteen patients with iNPH and 10 age-matched controls were recruited. QSI data were obtained with a 3-T system by using a single-shot echo planar imaging sequence with the diffusion gradient applied parallel to the antero-posterior axis. By using a two-component low-q fit model, the root mean square displacements of intra-axonal space ( =  axon diameter and intra-axonal volume fraction of the CST were calculated at the levels of the internal capsule and body of the lateral ventricle, respectively. RESULTS: Wilcoxon's rank-sum test revealed a significant increase in CST intra-axonal volume fraction at the paraventricular level in patients (p<0.001, whereas no significant difference was observed in the axon diameter. At the level of the internal capsule, neither axon diameter nor intra-axonal volume fraction differed significantly between the two groups. CONCLUSION: Our results suggest that in patients with iNPH, the CST does not undergo irreversible axonal damage but is rather compressed and/or stretched owing to pressure from the enlarged ventricle. These analyses of axon diameter and intra-axonal fraction yield insights into microstructural alterations of the CST in iNPH.

  16. Oxidative Damage Compromises Energy Metabolism in the Axonal Degeneration Mouse Model of X-Adrenoleukodystrophy (United States)

    Galino, Jorge; Ruiz, Montserrat; Fourcade, Stéphane; Schlüter, Agatha; López-Erauskin, Jone; Guilera, Cristina; Jove, Mariona; Naudi, Alba; García-Arumí, Elena; Andreu, Antoni L.; Starkov, Anatoly A.; Pamplona, Reinald; Ferrer, Isidre; Portero-Otin, Manuel


    Abstract Aims Chronic metabolic impairment and oxidative stress are associated with the pathogenesis of axonal dysfunction in a growing number of neurodegenerative conditions. To investigate the intertwining of both noxious factors, we have chosen the mouse model of adrenoleukodystrophy (X-ALD), which exhibits axonal degeneration in spinal cords and motor disability. The disease is caused by loss of function of the ABCD1 transporter, involved in the import and degradation of very long-chain fatty acids (VLCFA) in peroxisomes. Oxidative stress due to VLCFA excess appears early in the neurodegenerative cascade. Results In this study, we demonstrate by redox proteomics that oxidative damage to proteins specifically affects five key enzymes of glycolysis and TCA (Tricarboxylic acid) cycle in spinal cords of Abcd1− mice and pyruvate kinase in human X-ALD fibroblasts. We also show that NADH and ATP levels are significantly diminished in these samples, together with decrease of pyruvate kinase activities and GSH levels, and increase of NADPH. Innovation Treating Abcd1− mice with the antioxidants N-acetylcysteine and α-lipoic acid (LA) prevents protein oxidation; preserves NADH, NADPH, ATP, and GSH levels; and normalizes pyruvate kinase activity, which implies that oxidative stress provoked by VLCFA results in bioenergetic dysfunction, at a presymptomatic stage. Conclusion Our results provide mechanistic insight into the beneficial effects of antioxidants and enhance the rationale for translation into clinical trials for X-adrenoleukodystrophy. Antioxid. Redox Signal. 15, 2095–2107. PMID:21453200

  17. Pioglitazone halts axonal degeneration in a mouse model of X-linked adrenoleukodystrophy (United States)

    Morató, Laia; Galino, Jorge; Ruiz, Montserrat; Calingasan, Noel Ylagan; Starkov, Anatoly A.; Dumont, Magali; Naudí, Alba; Martínez, Juan José; Aubourg, Patrick; Portero-Otín, Manuel; Pamplona, Reinald; Galea, Elena; Beal, M. Flint; Ferrer, Isidre; Fourcade, Stéphane


    X-linked adrenoleukodystrophy is a neurometabolic disorder caused by inactivation of the peroxisomal ABCD1 transporter of very long-chain fatty acids. In mice, ABCD1 loss causes late onset axonal degeneration in the spinal cord in association with locomotor disability resembling the most common phenotype in patients, adrenomyeloneuropathy. Increasing evidence indicates that oxidative stress and bioenergetic failure play major roles in the pathogenesis of X-linked adrenoleukodystrophy. In this study, we aimed to evaluate whether mitochondrial biogenesis is affected in X-linked adrenoleukodystrophy. We demonstrated that Abcd1 null mice show reduced mitochondrial DNA concomitant with downregulation of mitochondrial biogenesis pathway driven by PGC-1α/PPARγ and reduced expression of mitochondrial proteins cytochrome c, NDUFB8 and VDAC. Moreover, we show that the oral administration of pioglitazone, an agonist of PPARγ, restored mitochondrial content and expression of master regulators of biogenesis, neutralized oxidative damage to proteins and DNA, and reversed bioenergetic failure in terms of ATP levels, NAD+/NADH ratios, pyruvate kinase and glutathione reductase activities. Most importantly, the treatment halted locomotor disability and axonal damage in X-linked adrenoleukodystrophy mice. These results lend support to the use of pioglitazone in clinical trials with patients with adrenomyeloneuropathy and reveal novel molecular mechanisms of action of pioglitazone in neurodegeneration. Future studies should address the effects of this anti-diabetic drug on other axonopathies in which oxidative stress and mitochondrial dysfunction are contributing factors. PMID:23794606

  18. Pioglitazone halts axonal degeneration in a mouse model of X-linked adrenoleukodystrophy. (United States)

    Morató, Laia; Galino, Jorge; Ruiz, Montserrat; Calingasan, Noel Ylagan; Starkov, Anatoly A; Dumont, Magali; Naudí, Alba; Martínez, Juan José; Aubourg, Patrick; Portero-Otín, Manuel; Pamplona, Reinald; Galea, Elena; Beal, M Flint; Ferrer, Isidre; Fourcade, Stéphane; Pujol, Aurora


    X-linked adrenoleukodystrophy is a neurometabolic disorder caused by inactivation of the peroxisomal ABCD1 transporter of very long-chain fatty acids. In mice, ABCD1 loss causes late onset axonal degeneration in the spinal cord in association with locomotor disability resembling the most common phenotype in patients, adrenomyeloneuropathy. Increasing evidence indicates that oxidative stress and bioenergetic failure play major roles in the pathogenesis of X-linked adrenoleukodystrophy. In this study, we aimed to evaluate whether mitochondrial biogenesis is affected in X-linked adrenoleukodystrophy. We demonstrated that Abcd1 null mice show reduced mitochondrial DNA concomitant with downregulation of mitochondrial biogenesis pathway driven by PGC-1α/PPARγ and reduced expression of mitochondrial proteins cytochrome c, NDUFB8 and VDAC. Moreover, we show that the oral administration of pioglitazone, an agonist of PPARγ, restored mitochondrial content and expression of master regulators of biogenesis, neutralized oxidative damage to proteins and DNA, and reversed bioenergetic failure in terms of ATP levels, NAD+/NADH ratios, pyruvate kinase and glutathione reductase activities. Most importantly, the treatment halted locomotor disability and axonal damage in X-linked adrenoleukodystrophy mice. These results lend support to the use of pioglitazone in clinical trials with patients with adrenomyeloneuropathy and reveal novel molecular mechanisms of action of pioglitazone in neurodegeneration. Future studies should address the effects of this anti-diabetic drug on other axonopathies in which oxidative stress and mitochondrial dysfunction are contributing factors.

  19. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

    DEFF Research Database (Denmark)

    Mann, Matthias; Ong, Shao En; Grønborg, Mads


    In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling...

  20. Membrane phosphorylation and nerve cell function

    International Nuclear Information System (INIS)

    Baer, P.R.


    This thesis deals with the phosphorylation of membrane components. In part I a series of experiments is described using the hippocampal slice as a model system. In part II a different model system - cultured hybrid cells - is used to study protein and lipid phosphorylation, influenced by incubation with neuropeptides. In part III in vivo and in vitro studies are combined to study protein phosphorylation after neuroanatomical lesions. In a section of part II (Page 81-90) labelling experiments of the membrane inositol-phospholipids are described. 32 P-ATP was used to label phospholipids in intact hybrid cells, and short incubations were found to be the most favourable. (C.F.)

  1. Characterizing the composition of molecular motors on moving axonal cargo using "cargo mapping" analysis. (United States)

    Neumann, Sylvia; Campbell, George E; Szpankowski, Lukasz; Goldstein, Lawrence S B; Encalada, Sandra E


    Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate ("map") the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. "Cargo mapping" consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to "map" them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for

  2. Difference in trafficking of brain-derived neurotrophic factor between axons and dendrites of cortical neurons, revealed by live-cell imaging

    Directory of Open Access Journals (Sweden)

    Kohara Keigo


    Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF, which is sorted into a regulated secretory pathway of neurons, is supposed to act retrogradely through dendrites on presynaptic neurons or anterogradely through axons on postsynaptic neurons. Depending on which is the case, the pattern and direction of trafficking of BDNF in dendrites and axons are expected to be different. To address this issue, we analyzed movements of green fluorescent protein (GFP-tagged BDNF in axons and dendrites of living cortical neurons by time-lapse imaging. In part of the experiments, the expression of BDNF tagged with cyan fluorescent protein (CFP was compared with that of nerve growth factor (NGF tagged with yellow fluorescent protein (YFP, to see whether fluorescent protein-tagged BDNF is expressed in a manner specific to this neurotrophin. Results We found that BDNF tagged with GFP or CFP was expressed in a punctated manner in dendrites and axons in about two-thirds of neurons into which plasmid cDNAs had been injected, while NGF tagged with GFP or YFP was diffusely expressed even in dendrites in about 70% of the plasmid-injected neurons. In neurons in which BDNF-GFP was expressed as vesicular puncta in axons, 59 and 23% of the puncta were moving rapidly in the anterograde and retrograde directions, respectively. On the other hand, 64% of BDNF-GFP puncta in dendrites did not move at all or fluttered back and forth within a short distance. The rest of the puncta in dendrites were moving relatively smoothly in either direction, but their mean velocity of transport, 0.47 ± 0.23 (SD μm/s, was slower than that of the moving puncta in axons (0.73 ± 0.26 μm/s. Conclusion The present results show that the pattern and velocity of the trafficking of fluorescence protein-tagged BDNF are different between axons and dendrites, and suggest that the anterograde transport in axons may be the dominant stream of BDNF to release sites.

  3. Multiple sclerosis and anterograde axonal degeneration study by magnetic resonance

    International Nuclear Information System (INIS)

    Martinez Pardo, P.; Capdevila Cirera, A.; Sanz Marin, P.M.; Gili Planas, J.


    Multiple sclerosis (MS) is a disease of the central nervous system that affects specifically the myelin. Its diagnosis by imaging techniques is, since the development of magnetic resonance (MR), relatively simple, and its occasional association with anterograde axonal degeneration (WD) has been reported. In both disorders, there is a lengthening of the T1 and T2 relaxation times. In the present report, 76 patients with MS with less than 4 plaques in the typical periventricular position were studied retrospectively, resulting in a rate of association with anterograde axonal degeneration of 8%. We consider that in spite of their same behavior in MR,MS and WD, with moreover represent completely different pathologies, are perfectly differential by MR. The S-E images with longer repetition and echo times in the axial and coronal planes have proved to be those most sensitive for this differentiation. Given that MS is specific pathology of then myelin, the axonal damages in delayed until several plaques adjacent to an axon affect it. We consider that this, added to the restriction of our study group (less than 4 plaques), is the cause of the pow percentage of the MS-WD association in our study. (Author)

  4. Model of fasciculation and sorting in mixed populations of axons

    Czech Academy of Sciences Publication Activity Database

    Chaudhuri, D.; Borowski, P.; Zápotocký, Martin


    Roč. 84, č. 2 (2011), e021908 ISSN 1539-3755 R&D Projects: GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : axon guidance * neurogenesis * mathematical model Subject RIV: FH - Neurology Impact factor: 2.255, year: 2011

  5. Axonal dynamics of excitatory and inhibitory neurons in somatosensory cortex.

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    Sally A Marik


    Full Text Available Cortical topography can be remapped as a consequence of sensory deprivation, suggesting that cortical circuits are continually modified by experience. To see the effect of altered sensory experience on specific components of cortical circuits, we imaged neurons, labeled with a genetically modified adeno-associated virus, in the intact mouse somatosensory cortex before and after whisker plucking. Following whisker plucking we observed massive and rapid reorganization of the axons of both excitatory and inhibitory neurons, accompanied by a transient increase in bouton density. For horizontally projecting axons of excitatory neurons there was a net increase in axonal projections from the non-deprived whisker barrel columns into the deprived barrel columns. The axon collaterals of inhibitory neurons located in the deprived whisker barrel columns retracted in the vicinity of their somata and sprouted long-range projections beyond their normal reach towards the non-deprived whisker barrel columns. These results suggest that alterations in the balance of excitation and inhibition in deprived and non-deprived barrel columns underlie the topographic remapping associated with sensory deprivation.

  6. Acute Motor Axonal Neuropathy in Association with Hepatitis E

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    Araz Al-Saffar


    Full Text Available Guillain–Barré syndrome (GBS is an acute peripheral neuropathy that develops as a result of post-infectious immune-mediated nerve injury. It can be classified into classic and variant GBS. Acute motor axonal neuropathy (AMAN is a subtype of GBS with the key clinical features of pure motor weakness, areflexia, absence of sensory symptoms, and lack of neurophysiologic evidence of demyelination. We reported a case of acute motor axonal neuropathy in association with hepatitis E infection. A young woman was referred to us after a period of nausea, fever, and diarrhea. She had unexplained muscle weakness at admission and has been diagnosed with acute hepatitis E infection. A rigorous clinical neurological assessment revealed bilateral symmetrical weakness, which affects the lower limbs more than the upper limbs, with no evidence of sensory involvement. Neurophysiological measurements indicated acute axonal injury without clues to demyelination. A diagnosis of acute motor axonal neuropathy subtype has been made, to which she only received supportive therapy. The symptoms resolved spontaneously and full recovery of motor function was attained after 35 days of weakness onset with complete normalization of neurophysiologic parameters.

  7. Investigation on the mechanism of peripheral axonal injury in glaucoma

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    Jun- Hong Zhao


    Full Text Available AIM: To compare the angles of longitudinal section of sclera around optic nerve heads and the never fiber layer changes in healthy adults and patients with glaucoma, and to investigate the mechanism of peripheral retinal axonal injury, with the combined knowledge of biomechanics. METHODS: The optical nerves and their peripheral tissue specimen in the 12 eyes from health adult donators and 12 eyes from glaucoma patient donators were dyed by Glees' method to compare the angles of longitudinal section of sclera around optic nerve heads(through optic nerve center, and to observe the anatomical features of the peripheral retinal axons. RESULTS: The mean angle of longitudinal section of sclera around optic nerve in healthy adults was 73.3°, while that in patients with absolute glaucoma was 75.6°. The difference showed no significance(t=1.44, P>0.05. There was a sharp bend in the course of peripheral optical fiber in healthy adults. However, the optic nerve fiber disappeared completely in patients with glaucoma end stage. CONCLUSION: The angle between the medial edge and leading edge of sclera(around optic nerve headsis an acute angle. The optical fiber in glaucoma end stage disappeared completely. The phenomenon may be related to high intraocular pressure, the sclera shape, the shear modulus of sclera and axons, and “axonal bending-injury” mechanism.

  8. IFNgamma enhances microglial reactions to hippocampal axonal degeneration

    DEFF Research Database (Denmark)

    Jensen, M B; Hegelund, I V; Lomholt, N D


    Glial reactivity is implicated in CNS repair and regenerative responses. Microglia, the cells responding earliest to axonal injury, produce tumor necrosis factor-alpha (TNFalpha), a cytokine with both cytopathic and neuroprotective effects. We have studied activation of hippocampal microglia to p...

  9. Differential Axonal Projection of Mitral and Tufted Cells in the Mouse Main Olfactory System

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    Shin Nagayama


    Full Text Available In the past decade, much has been elucidated regarding the functional organization of the axonal connection of olfactory sensory neurons to olfactory bulb (OB glomeruli. However, the manner in which projection neurons of the OB process odorant input and send this information to higher brain centers remains unclear. Here, we report long-range, large-scale tracing of the axonal projection patterns of OB neurons using two-photon microscopy. Tracer injection into a single glomerulus demonstrated widely distributed mitral/tufted cell axonal projections on the lateroventral surface of the mouse brain, including the anterior/posterior piriform cortex (PC and olfactory tubercle (OT. We noted two distinct groups of labeled axons: PC-orienting axons and OT-orienting axons. Each group occupied distinct parts of the lateral olfactory tract. PC-orienting axons projected axon collaterals to a wide area of the PC but only a few collaterals to the OT. OT-orienting axons densely projected axon collaterals primarily to the anterolateral OT (alOT. Different colored dye injections into the superficial and deep portions of the OB external plexiform layer revealed that the PC-orienting axon populations originated in presumed mitral cells and the OT-orienting axons in presumed tufted cells. These data suggest that although mitral and tufted cells receive similar odor signals from a shared glomerulus, they process the odor information in different ways and send their output to different higher brain centers via the PC and alOT.

  10. Dynamics of survival of motor neuron (SMN) protein interaction with the mRNA-binding protein IMP1 facilitates its trafficking into motor neuron axons. (United States)

    Fallini, Claudia; Rouanet, Jeremy P; Donlin-Asp, Paul G; Guo, Peng; Zhang, Honglai; Singer, Robert H; Rossoll, Wilfried; Bassell, Gary J


    Spinal muscular atrophy (SMA) is a lethal neurodegenerative disease specifically affecting spinal motor neurons. SMA is caused by the homozygous deletion or mutation of the survival of motor neuron 1 (SMN1) gene. The SMN protein plays an essential role in the assembly of spliceosomal ribonucleoproteins. However, it is still unclear how low levels of the ubiquitously expressed SMN protein lead to the selective degeneration of motor neurons. An additional role for SMN in the regulation of the axonal transport of mRNA-binding proteins (mRBPs) and their target mRNAs has been proposed. Indeed, several mRBPs have been shown to interact with SMN, and the axonal levels of few mRNAs, such as the β-actin mRNA, are reduced in SMA motor neurons. In this study we have identified the β-actin mRNA-binding protein IMP1/ZBP1 as a novel SMN-interacting protein. Using a combination of biochemical assays and quantitative imaging techniques in primary motor neurons, we show that IMP1 associates with SMN in individual granules that are actively transported in motor neuron axons. Furthermore, we demonstrate that IMP1 axonal localization depends on SMN levels, and that SMN deficiency in SMA motor neurons leads to a dramatic reduction of IMP1 protein levels. In contrast, no difference in IMP1 protein levels was detected in whole brain lysates from SMA mice, further suggesting neuron specific roles of SMN in IMP1 expression and localization. Taken together, our data support a role for SMN in the regulation of mRNA localization and axonal transport through its interaction with mRBPs such as IMP1. Copyright © 2013 Wiley Periodicals, Inc.

  11. Mapping of p140Cap phosphorylation sites

    DEFF Research Database (Denmark)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta


    Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes...

  12. Genetic Deletion of the Transcriptional Repressor NFIL3 Enhances Axon Growth In Vitro but Not Axonal Repair In Vivo

    NARCIS (Netherlands)

    van der Kallen, Loek R; Eggers, Ruben; Ehlert, Erich M; Verhaagen, J.; Smit, August B; van Kesteren, Ronald E


    Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes Arg1 and Gap43 and strongly

  13. Axonal regeneration and development of de novo axons from distal dendrites of adult feline commissural interneurons after a proximal axotomy

    DEFF Research Database (Denmark)

    Fenrich, Keith K; Skelton, Nicole; MacDermid, Victoria E


    Following proximal axotomy, several types of neurons sprout de novo axons from distal dendrites. These processes may represent a means of forming new circuits following spinal cord injury. However, it is not know whether mammalian spinal interneurons, axotomized as a result of a spinal cord injur...

  14. Oligodendrocyte Development in the Absence of Their Target Axons In Vivo.

    Directory of Open Access Journals (Sweden)

    Rafael Almeida

    Full Text Available Oligodendrocytes form myelin around axons of the central nervous system, enabling saltatory conduction. Recent work has established that axons can regulate certain aspects of oligodendrocyte development and myelination, yet remarkably oligodendrocytes in culture retain the ability to differentiate in the absence of axons and elaborate myelin sheaths around synthetic axon-like substrates. It remains unclear the extent to which the life-course of oligodendrocytes requires the presence of, or signals derived from axons in vivo. In particular, it is unclear whether the specific axons fated for myelination regulate the oligodendrocyte population in a living organism, and if so, which precise steps of oligodendrocyte-cell lineage progression are regulated by target axons. Here, we use live-imaging of zebrafish larvae carrying transgenic reporters that label oligodendrocyte-lineage cells to investigate which aspects of oligodendrocyte development, from specification to differentiation, are affected when we manipulate the target axonal environment. To drastically reduce the number of axons targeted for myelination, we use a previously identified kinesin-binding protein (kbp mutant, in which the first myelinated axons in the spinal cord, reticulospinal axons, do not fully grow in length, creating a region in the posterior spinal cord where most initial targets for myelination are absent. We find that a 73% reduction of reticulospinal axon surface in the posterior spinal cord of kbp mutants results in a 27% reduction in the number of oligodendrocytes. By time-lapse analysis of transgenic OPC reporters, we find that the reduction in oligodendrocyte number is explained by a reduction in OPC proliferation and survival. Interestingly, OPC specification and migration are unaltered in the near absence of normal axonal targets. Finally, we find that timely differentiation of OPCs into oligodendrocytes does not depend at all on the presence of target axons

  15. Adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) and Nasu-Hakola disease: lesion staging and dynamic changes of axons and microglial subsets. (United States)

    Oyanagi, Kiyomitsu; Kinoshita, Michiaki; Suzuki-Kouyama, Emi; Inoue, Teruhiko; Nakahara, Asa; Tokiwai, Mika; Arai, Nobutaka; Satoh, Jun-Ichi; Aoki, Naoya; Jinnai, Kenji; Yazawa, Ikuru; Arai, Kimihito; Ishihara, Kenji; Kawamura, Mitsuru; Ishizawa, Keisuke; Hasegawa, Kazuko; Yagisita, Saburo; Amano, Naoji; Yoshida, Kunihiro; Terada, Seishi; Yoshida, Mari; Akiyama, Haruhiko; Mitsuyama, Yoshio; Ikeda, Shu-Ichi


    The brains of 10 Japanese patients with adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) encompassing hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) and pigmentary orthochromatic leukodystrophy (POLD) and eight Japanese patients with Nasu-Hakola disease (N-HD) and five age-matched Japanese controls were examined neuropathologically with special reference to lesion staging and dynamic changes of microglial subsets. In both diseases, the pathognomonic neuropathological features included spherically swollen axons (spheroids and globules), axon loss and changes of microglia in the white matter. In ALSP, four lesion stages based on the degree of axon loss were discernible: Stage I, patchy axon loss in the cerebral white matter without atrophy; Stage II, large patchy areas of axon loss with slight atrophy of the cerebral white matter and slight dilatation of the lateral ventricles; Stage III, extensive axon loss in the cerebral white matter and dilatation of the lateral and third ventricles without remarkable axon loss in the brainstem and cerebellum; Stage IV, devastated cerebral white matter with marked dilatation of the ventricles and axon loss in the brainstem and/or cerebellum. Internal capsule and pontine base were relatively well preserved in the N-HD, even at Stage IV, and the swollen axons were larger with a higher density in the ALSP. Microglial cells immunopositive for CD68, CD163 or CD204 were far more obvious in ALSP, than in N-HD, and the shape and density of the cells changed in each stage. With progression of the stage, clinical symptoms became worse to apathetic state, and epilepsy was frequently observed in patients at Stages III and IV in both diseases. From these findings, it is concluded that (i) shape, density and subsets of microglia change dynamically along the passage of stages and (ii) increase of IBA-1-, CD68-, CD163- and CD204-immunopositive cells precedes loss of axons in ALSP. © 2016

  16. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    International Nuclear Information System (INIS)

    Marasini, Carlotta; Galeno, Lauretta; Moran, Oscar


    Highlights: ► CFTR mutations produce cystic fibrosis. ► Chloride transport depends on the regulatory domain phosphorylation. ► Regulatory domain is intrinsically disordered. ► Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and β-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of α-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two conditions, monitoring the changes of the mean residue ellipticity measured at 222 nm as a function of temperature

  17. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    Energy Technology Data Exchange (ETDEWEB)

    Marasini, Carlotta, E-mail: [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy); Galeno, Lauretta; Moran, Oscar [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy)


    Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

  18. An αII Spectrin-Based Cytoskeleton Protects Large-Diameter Myelinated Axons from Degeneration. (United States)

    Huang, Claire Yu-Mei; Zhang, Chuansheng; Zollinger, Daniel R; Leterrier, Christophe; Rasband, Matthew N


    Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with βIV and βII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1 f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K + channels. We show that the density of nodal βIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1 f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier. SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in

  19. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    International Nuclear Information System (INIS)

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon


    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

  20. Anterograde glycoprotein-dependent transport of newly generated rabies virus in dorsal root ganglion neurons. (United States)

    Bauer, Anja; Nolden, Tobias; Schröter, Josephine; Römer-Oberdörfer, Angela; Gluska, Shani; Perlson, Eran; Finke, Stefan


    Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of

  1. International symposium on cellular and molecular biology of phosphate and phosphorylated compounds in microorganisms: Proceedings

    Energy Technology Data Exchange (ETDEWEB)



    This report contains the abstracts of papers presented at the conference. Attention is focused on the following topics: regulation of phosphate metabolism in bacteria; structure-function of alkaline phosphatase; regulation of phosphate metabolism in yeast; transport of phosphate and phosphorylated compounds; and phosphate regulation in pathogenesis and secondary metabolism.

  2. Autophagy induction halts axonal degeneration in a mouse model of X-adrenoleukodystrophy. (United States)

    Launay, Nathalie; Aguado, Carmen; Fourcade, Stéphane; Ruiz, Montserrat; Grau, Laia; Riera, Jordi; Guilera, Cristina; Giròs, Marisa; Ferrer, Isidre; Knecht, Erwin; Pujol, Aurora


    X-linked adrenoleukodystrophy (X-ALD) is a rare neurometabolic disease characterized by the accumulation of very long chain fatty acids (VLCFAs) due to a loss of function of the peroxisomal transporter ABCD1. Here, using in vivo and in vitro models, we demonstrate that autophagic flux was impaired due to elevated mammalian target of rapamycin (mTOR) signaling, which contributed to X-ALD pathogenesis. We also show that excess VLCFAs downregulated autophagy in human fibroblasts. Furthermore, mTOR inhibition by a rapamycin derivative (temsirolimus) restored autophagic flux and inhibited the axonal degenerative process as well as the associated locomotor impairment in the Abcd1 (-) /Abcd2 (-/-) mouse model. This process was mediated through the restoration of proteasome function and redox as well as metabolic homeostasis. These findings provide the first evidence that links impaired autophagy to X-ALD, which may yield a therapy based on autophagy activators for adrenomyeloneuropathy patients.

  3. Hemin inhibits internalization of transferrin by reticulocytes and promotes phosphorylation of the membrane transferrin receptor

    International Nuclear Information System (INIS)

    Cox, T.M.; O'Donnell, M.W.; Aisen, P.; London, I.M.


    Addition of hemin to reticulocytes inhibits incorporation of iron from transferrin. Heme also regulates protein synthesis in immature erythroid cells through its effects on phosphorylation of the initiation factor eIF-2. The authors have examined its effects on endocytosis of iron-transferrin and phosphorylation of the transferrin receptor. Hemin reduced iron transport but increased cell-associated transferrin. During uptake of 125 I-labeled transferrin in the steady state, the use of a washing technique to dissociate bound transferrin on the cell membrane showed that radioligand accumulated on the surface of hemin-treated cells. Receptor phosphorylation was investigated by immunoprecipitation of reticulocyte extracts after metabolic labeling with [ 32 P]P/sub i/. In the absence of ligand, phosphorylated receptor was chiefly localized on cell stroma. Exposure to transferrin increased cytosolic phosphorylated receptor from 15-30% to approximately 50% of the total, an effect overcome by hemin treatment. The findings suggest a possible relationship of phosphorylation to endocytosis of the transferrin receptor in reticulocytes

  4. Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+. (United States)

    Villalobo, A; Lehninger, A L


    Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

  5. Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner. (United States)

    Mitew, Stanislaw; Gobius, Ilan; Fenlon, Laura R; McDougall, Stuart J; Hawkes, David; Xing, Yao Lulu; Bujalka, Helena; Gundlach, Andrew L; Richards, Linda J; Kilpatrick, Trevor J; Merson, Tobias D; Emery, Ben


    Mounting evidence suggests that neuronal activity influences myelination, potentially allowing for experience-driven modulation of neural circuitry. The degree to which neuronal activity is capable of regulating myelination at the individual axon level is unclear. Here we demonstrate that stimulation of somatosensory axons in the mouse brain increases proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) within the underlying white matter. Stimulated axons display an increased probability of being myelinated compared to neighboring non-stimulated axons, in addition to being ensheathed with thicker myelin. Conversely, attenuating neuronal firing reduces axonal myelination in a selective activity-dependent manner. Our findings reveal that the process of selecting axons for myelination is strongly influenced by the relative activity of individual axons within a population. These observed cellular changes are consistent with the emerging concept that adaptive myelination is a key mechanism for the fine-tuning of neuronal circuitry in the mammalian CNS.

  6. Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals. (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi


    The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

  7. The redox state and the phosphorylation state of the mannitol-specific carrier of the E. coli phosphoenolpyruvate-dependent phosphotransferase system

    NARCIS (Netherlands)

    Robillard, G.T.; Pas, H.H.; Gage, D.; Elferink, M.G.L.


    This review summarizes the recent developments in identifying the activity-linked cysteine as one of the phosphorylation sites on the mannitol-specific EII of the E. coli phosphoenolpyruvate-dependent mannitol transport system. Two phosphorylation sites have been identified, one being the HPr/P-HPr

  8. A growing field: The regulation of axonal regeneration by Wnt signaling. (United States)

    Garcia, Armando L; Udeh, Adanna; Kalahasty, Karthik; Hackam, Abigail S


    The canonical Wnt/β-catenin pathway is a highly conserved signaling cascade that plays critical roles during embryogenesis. Wnt ligands regulate axonal extension, growth cone guidance and synaptogenesis throughout the developing central nervous system (CNS). Recently, studies in mammalian and fish model systems have demonstrated that Wnt/β-catenin signaling also promotes axonal regeneration in the adult optic nerve and spinal cord after injury, raising the possibility that Wnt could be developed as a therapeutic strategy. In this review, we summarize experimental evidence that reveals novel roles for Wnt signaling in the injured CNS, and discuss possible mechanisms by which Wnt ligands could overcome molecular barriers inhibiting axonal growth to promote regeneration. A central challenge in the neuroscience field is developing therapeutic strategies that induce robust axonal regeneration. Although adult axons have the capacity to respond to axonal guidance molecules after injury, there are several major obstacles for axonal growth, including extensive neuronal death, glial scars at the injury site, and lack of axonal guidance signals. Research in rodents demonstrated that activation of Wnt/β-catenin signaling in retinal neurons and radial glia induced neuronal survival and axonal growth, but that activation within reactive glia at the injury site promoted proliferation and glial scar formation. Studies in zebrafish spinal cord injury models confirm an axonal regenerative role for Wnt/β-catenin signaling and identified the cell types responsible. Additionally, in vitro and in vivo studies demonstrated that Wnt induces axonal and neurite growth through transcription-dependent effects of its central mediator β-catenin, potentially by inducing regeneration-promoting genes. Canonical Wnt signaling may also function through transcription-independent interactions of β-catenin with cytoskeletal elements, which could stabilize growing axons and control growth cone

  9. Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins. (United States)

    Gainer, Harold; House, Shirley; Kim, Dong Sun; Chin, Hemin; Pant, Harish C


    When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia-axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

  10. Neocortical axon arbors trade-off material and conduction delay conservation.

    Directory of Open Access Journals (Sweden)

    Julian M L Budd


    Full Text Available The brain contains a complex network of axons rapidly communicating information between billions of synaptically connected neurons. The morphology of individual axons, therefore, defines the course of information flow within the brain. More than a century ago, Ramón y Cajal proposed that conservation laws to save material (wire length and limit conduction delay regulate the design of individual axon arbors in cerebral cortex. Yet the spatial and temporal communication costs of single neocortical axons remain undefined. Here, using reconstructions of in vivo labelled excitatory spiny cell and inhibitory basket cell intracortical axons combined with a variety of graph optimization algorithms, we empirically investigated Cajal's conservation laws in cerebral cortex for whole three-dimensional (3D axon arbors, to our knowledge the first study of its kind. We found intracortical axons were significantly longer than optimal. The temporal cost of cortical axons was also suboptimal though far superior to wire-minimized arbors. We discovered that cortical axon branching appears to promote a low temporal dispersion of axonal latencies and a tight relationship between cortical distance and axonal latency. In addition, inhibitory basket cell axonal latencies may occur within a much narrower temporal window than excitatory spiny cell axons, which may help boost signal detection. Thus, to optimize neuronal network communication we find that a modest excess of axonal wire is traded-off to enhance arbor temporal economy and precision. Our results offer insight into the principles of brain organization and communication in and development of grey matter, where temporal precision is a crucial prerequisite for coincidence detection, synchronization and rapid network oscillations.

  11. Coevolution of axon guidance molecule Slit and its receptor Robo.

    Directory of Open Access Journals (Sweden)

    Qi Yu

    Full Text Available Coevolution is important for the maintenance of the interaction between a ligand and its receptor during evolution. The interaction between axon guidance molecule Slit and its receptor Robo is critical for the axon repulsion in neural tissues, which is evolutionarily conserved from planarians to humans. However, the mechanism of coevolution between Slit and Robo remains unclear. In this study, we found that coordinated amino acid changes took place at interacting sites of Slit and Robo by comparing the amino acids at these sites among different organisms. In addition, the high level correlation between evolutionary rate of Slit and Robo was identified in vertebrates. Furthermore, the sites under positive selection of slit and robo were detected in the same lineage such as mosquito and teleost. Overall, our results provide evidence for the coevolution between Slit and Robo.

  12. Regulation of gap junctions by protein phosphorylation

    Directory of Open Access Journals (Sweden)

    J.C. Sáez


    Full Text Available Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

  13. Activated retinal glia mediated axon regeneration in experimental glaucoma. (United States)

    Lorber, Barbara; Guidi, Alessandra; Fawcett, James W; Martin, Keith R


    Glaucoma, a leading cause of blindness, is a neurodegenerative disease characterized by progressive loss of retinal ganglion cell axons in the optic nerve and their cell bodies in the retina. Reactive retinal glial changes have been observed in glaucoma but the role of such glial changes in the pathogenesis of the condition remains unclear. In the present study we found that retinal ganglion cells in an experimental animal model of glaucoma have an increased axon regenerative potential. Regeneration of adult rat retinal ganglion cell axons after optic nerve crush was significantly increased in vivo when combined with intraocular pressure-induced experimental glaucoma. This enhanced axon regeneration response was correlated with a significant increase in activation of glial fibrillary acidic protein+retinal glia. Using a dissociated retinal ganglion cell culture model we showed that reducing the number of activated retinal glia with a glial specific toxin, α-Aminoadipic acid, significantly reduced the growth potential of retinal ganglion cells from glaucomatous rat eyes, suggesting that activated retinal glia mediate, at least in part, the growth promoting effect. This was shown to be mediated by both membrane-bound and soluble glial-derived factors. Neurotrophin and ciliary neurotrophic/leukemia inhibitory factor blockers did not affect the regenerative potential, excluding these growth factors as principal mediators of the enhanced growth response occurring in glaucomatous retinal cultures. These observations are the first to reveal that retinal ganglion cells from glaucomatous rat eyes have an enhanced regenerative capacity. Furthermore, our results suggest that activated retinal glia mediate at least part of this response. Further work to understand and enhance the regeneration-promoting effect of activated retinal glia is required to determine if this approach could be useful as part of a therapeutic strategy to encourage optic nerve regeneration in glaucoma

  14. [Craniocerebral trauma: magnetic resonance imaging of diffuse axonal injury]. (United States)

    Mallouhi, A


    Acceleration-deceleration rotational brain trauma is a common cause of disability or death in young adults and often leads to a focal destruction of axons. The resulting pathology, axonal shear injury is referred to as diffuse axonal injury (DAI). The DAI-associated lesions occur bilaterally, are widely dispersed and have been observed in the surface and deep white matter. They are found near to and far from the impact site. When DAI is clinically suspected, magnetic resonance imaging (MRI) is the method of choice for further clarification, especially in patients where cranial computed tomography (CT) is inconspicuous. To investigate the presence of DAI after traumatic brain injury (TBI), a multimodal MRI approach is applied including the common structural and also functional imaging sequences. For structural MRI, fluid-attenuated inversion recovery (FLAIR) weighted and susceptibility contrast imaging (SWI) are the sequences mainly used. The SWI technique is extremely sensitive to blood breakdown products, which appear as small signal voids at three locations, at the gray-white interface, in the corpus callosum and in the brain stem. Functional MRI comprises a group of constantly developing techniques that have great potential in optimal evaluation of the white matter in patients after craniocerebral trauma. These imaging techniques allow the visualization of changes associated with shear injuries, such as functional impairment of axons and decreased blood flow and abnormal metabolic activity of the brain parts affected. The multimodal MRI approach in patients with DAI results in a more detailed and differentiated representation of the underlying pathophysiological changes of the injured nerve tracts and helps to improve the diagnostic and prognostic accuracy of MRI. When DAI is suspected multimodal MRI should be performed as soon as possible after craniocerebral injury.

  15. Spontaneous axonal regeneration in rodent spinal cord after ischemic injury

    DEFF Research Database (Denmark)

    von Euler, Mia; Janson, A M; Larsen, Jytte Overgaard


    cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord...... lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury....

  16. Polyethylene glycol restores axonal conduction after corpus callosum transection. (United States)

    Bamba, Ravinder; Riley, D Colton; Boyer, Richard B; Pollins, Alonda C; Shack, R Bruce; Thayer, Wesley P


    Polyethylene glycol (PEG) has been shown to restore axonal continuity after peripheral nerve transection in animal models. We hypothesized that PEG can also restore axonal continuity in the central nervous system. In this current experiment, coronal sectioning of the brains of Sprague-Dawley rats was performed after animal sacrifice. 3Brain high-resolution microelectrode arrays (MEA) were used to measure mean firing rate (MFR) and peak amplitude across the corpus callosum of the ex-vivo brain slices. The corpus callosum was subsequently transected and repeated measurements were performed. The cut ends of the corpus callosum were still apposite at this time. A PEG solution was applied to the injury site and repeated measurements were performed. MEA measurements showed that PEG was capable of restoring electrophysiology signaling after transection of central nerves. Before injury, the average MFRs at the ipsilateral, midline, and contralateral corpus callosum were 0.76, 0.66, and 0.65 spikes/second, respectively, and the average peak amplitudes were 69.79, 58.68, and 49.60 μV, respectively. After injury, the average MFRs were 0.71, 0.14, and 0.25 spikes/second, respectively and peak amplitudes were 52.11, 8.98, and 16.09 μV, respectively. After application of PEG, there were spikes in MFR and peak amplitude at the injury site and contralaterally. The average MFRs were 0.75, 0.55, and 0.47 spikes/second at the ipsilateral, midline, and contralateral corpus callosum, respectively and peak amplitudes were 59.44, 45.33, 40.02 μV, respectively. There were statistically differences in the average MFRs and peak amplitudes between the midline and non-midline corpus callosum groups ( P < 0.01, P < 0.05). These findings suggest that PEG restores axonal conduction between severed central nerves, potentially representing axonal fusion.

  17. Polyethylene glycol restores axonal conduction after corpus callosum transection

    Directory of Open Access Journals (Sweden)

    Ravinder Bamba


    Full Text Available Polyethylene glycol (PEG has been shown to restore axonal continuity after peripheral nerve transection in animal models. We hypothesized that PEG can also restore axonal continuity in the central nervous system. In this current experiment, coronal sectioning of the brains of Sprague-Dawley rats was performed after animal sacrifice. 3Brain high-resolution microelectrode arrays (MEA were used to measure mean firing rate (MFR and peak amplitude across the corpus callosum of the ex-vivo brain slices. The corpus callosum was subsequently transected and repeated measurements were performed. The cut ends of the corpus callosum were still apposite at this time. A PEG solution was applied to the injury site and repeated measurements were performed. MEA measurements showed that PEG was capable of restoring electrophysiology signaling after transection of central nerves. Before injury, the average MFRs at the ipsilateral, midline, and contralateral corpus callosum were 0.76, 0.66, and 0.65 spikes/second, respectively, and the average peak amplitudes were 69.79, 58.68, and 49.60 μV, respectively. After injury, the average MFRs were 0.71, 0.14, and 0.25 spikes/second, respectively and peak amplitudes were 52.11, 8.98, and 16.09 μV, respectively. After application of PEG, there were spikes in MFR and peak amplitude at the injury site and contralaterally. The average MFRs were 0.75, 0.55, and 0.47 spikes/second at the ipsilateral, midline, and contralateral corpus callosum, respectively and peak amplitudes were 59.44, 45.33, 40.02 μV, respectively. There were statistically differences in the average MFRs and peak amplitudes between the midline and non-midline corpus callosum groups (P < 0.01, P < 0.05. These findings suggest that PEG restores axonal conduction between severed central nerves, potentially representing axonal fusion.

  18. Two stable steady states in the Hodgkin-Huxley axons


    Aihara, K.; Matsumoto, G.


    Two stable steady states were found in the numerical solution of the Hodgkin-Huxley equations for the intact squid axon bathed in potassium-rich sea water with an externally applied inward current. Under the conditions the two stable steady-states exist, the Hodgkin-Huxley equations have a complex bifurcation structure including, in addition to the two stable steady-states, a stable limit cycle, two unstable equilibrium points, and one asymptotically stable equilibrium point. It was also conc...

  19. Dendritic and Axonal Wiring Optimization of Cortical GABAergic Interneurons. (United States)

    Anton-Sanchez, Laura; Bielza, Concha; Benavides-Piccione, Ruth; DeFelipe, Javier; Larrañaga, Pedro


    The way in which a neuronal tree expands plays an important role in its functional and computational characteristics. We aimed to study the existence of an optimal neuronal design for different types of cortical GABAergic neurons. To do this, we hypothesized that both the axonal and dendritic trees of individual neurons optimize brain connectivity in terms of wiring length. We took the branching points of real three-dimensional neuronal reconstructions of the axonal and dendritic trees of different types of cortical interneurons and searched for the minimal wiring arborization structure that respects the branching points. We compared the minimal wiring arborization with real axonal and dendritic trees. We tested this optimization problem using a new approach based on graph theory and evolutionary computation techniques. We concluded that neuronal wiring is near-optimal in most of the tested neurons, although the wiring length of dendritic trees is generally nearer to the optimum. Therefore, wiring economy is related to the way in which neuronal arborizations grow irrespective of the marked differences in the morphology of the examined interneurons.

  20. Prediction of Functional Outcome in Axonal Guillain-Barre Syndrome. (United States)

    Sung, Eun Jung; Kim, Dae Yul; Chang, Min Cheol; Ko, Eun Jae


    To identify the factors that could predict the functional outcome in patients with the axonal type of Guillain-Barre syndrome (GBS). Two hundred and two GBS patients admitted to our university hospital between 2003 and 2014 were reviewed retrospectively. We defined a good outcome as being "able to walk independently at 1 month after onset" and a poor outcome as being "unable to walk independently at 1 month after onset". We evaluated the factors that differed between the good and poor outcome groups. Twenty-four patients were classified into the acute motor axonal neuropathy type. There was a statistically significant difference between the good and poor outcome groups in terms of the GBS disability score at admission, and GBS disability score and Medical Research Council sum score at 1 month after admission. In an electrophysiologic analysis, the good outcome group showed greater amplitude of median, ulnar, deep peroneal, and posterior tibial nerve compound muscle action potentials (CMAP) and greater amplitude of median, ulnar, and superficial peroneal sensory nerve action potentials (SNAP) than the poor outcome group. A lower GBS disability score at admission, high amplitude of median, ulnar, deep peroneal, and posterior tibial CMAPs, and high amplitude of median, ulnar, and superficial peroneal SNAPs were associated with being able to walk at 1 month in patients with axonal GBS.

  1. Chlamydia trachomatis serovar L2 induces protein tyrosine phosphorylation during uptake by HeLa cells

    DEFF Research Database (Denmark)

    Birkelund, Svend; Johnsen, H; Christiansen, Gunna


    . By use of a monoclonal antibody against phosphotyrosine, we showed that three classes of proteins are tyrosine phosphorylated: a triple band of 68, 66, and 64 kDa, a 97-kDa band, and a 140-kDa band. The phosphorylation could be detected by immunoblotting from 15 min after infection of HeLa cells. We...... inactive. Attachment of EBs to host cells is medicated by a heparan sulfate-like glycosaminoglycan. Following attachment, the EB is internalized within a membrane-bound vesicle, and during the first 8 h of infection the vesicles are transported to a perinuclear location where they aggregate and fuse...

  2. Sodium Channel β2 Subunits Prevent Action Potential Propagation Failures at Axonal Branch Points. (United States)

    Cho, In Ha; Panzera, Lauren C; Chin, Morven; Hoppa, Michael B


    Neurotransmitter release depends on voltage-gated Na + channels (Na v s) to propagate an action potential (AP) successfully from the axon hillock to a synaptic terminal. Unmyelinated sections of axon are very diverse structures encompassing branch points and numerous presynaptic terminals with undefined molecular partners of Na + channels. Using optical recordings of Ca 2+ and membrane voltage, we demonstrate here that Na + channel β2 subunits (Na v β2s) are required to prevent AP propagation failures across the axonal arborization of cultured rat hippocampal neurons (mixed male and female). When Na v β2 expression was reduced, we identified two specific phenotypes: (1) membrane excitability and AP-evoked Ca 2+ entry were impaired at synapses and (2) AP propagation was severely compromised with >40% of axonal branches no longer responding to AP-stimulation. We went on to show that a great deal of electrical signaling heterogeneity exists in AP waveforms across the axonal arborization independent of axon morphology. Therefore, Na v β2 is a critical regulator of axonal excitability and synaptic function in unmyelinated axons. SIGNIFICANCE STATEMENT Voltage-gated Ca 2+ channels are fulcrums of neurotransmission that convert electrical inputs into chemical outputs in the form of vesicle fusion at synaptic terminals. However, the role of the electrical signal, the presynaptic action potential (AP), in modulating synaptic transmission is less clear. What is the fidelity of a propagating AP waveform in the axon and what molecules shape it throughout the axonal arborization? Our work identifies several new features of AP propagation in unmyelinated axons: (1) branches of a single axonal arborization have variable AP waveforms independent of morphology, (2) Na + channel β2 subunits modulate AP-evoked Ca 2+ -influx, and (3) β2 subunits maintain successful AP propagation across the axonal arbor. These findings are relevant to understanding the flow of excitation in the

  3. Dityrosine formation is impaired by tyrosine phosphorylation. (United States)

    Christian, S; Bernhard, G; Patrizia, R; Brigitte, M


    Using pure tyrosine and phosphotyrosine we have recently shown that phosphotyrosine is unable to form peroxidase catalyzed dimers (1989, FEBS Lett. 255, 395-397). In the present report, the effect of phosphotyrosine residues within a protein structure on dityrosine formation was studied using casein as a model protein. Dephosphorylation of casein resulted in a dose and time dependent increased synthesis of dityrosines following treatment with peroxidase/H2O2. The extent of crosslink formation was inversely related to the amount of phosphorylated tyrosine residues as quantitated by immunoblotting. Thus, phosphorylation of tyrosine residues could play a regulatory role in protein-crosslinking where dityrosine bonds are involved.

  4. Aeromedical evacuation-relevant hypobaria worsens axonal and neurologic injury in rats after underbody blast-induced hyperacceleration. (United States)

    Proctor, Julie L; Mello, Kaitlin T; Fang, Raymond; Puche, Adam C; Rosenthal, Robert E; Fourney, William L; Leiste, Ulrich H; Fiskum, Gary


    Occupants of military vehicles targeted by explosive devices often suffer from traumatic brain injury (TBI) and are typically transported by the aeromedical evacuation (AE) system to a military medical center within a few days. This study tested the hypothesis that exposure of rats to AE-relevant hypobaria worsens cerebral axonal injury and neurologic impairment caused by underbody blasts. Anesthetized adult male rats were secured within cylinders attached to a metal plate, simulating the hull of an armored vehicle. An explosive located under the plate was detonated, resulting in a peak vertical acceleration force on the plate and occupant rats of 100G. Rats remained under normobaria or were exposed to hypobaria equal to 8,000 feet in an altitude chamber for 6 hours, starting at 6 hours to 6 days after blast. At 7 days, rats were tested for vestibulomotor function using the balance beam walking task and euthanized by perfusion. The brains were then analyzed for axonal fiber injury. The number of internal capsule silver-stained axonal fibers was greater in animals exposed to 100G blast than in shams. Animals exposed to hypobaria starting at 6 hours to 6 days after blast exhibited more silver-stained fibers than those not exposed to hypobaria. Rats exposed to 100% oxygen (O2) during hypobaria at 24 hours postblast displayed greater silver staining and more balance beam foot-faults, in comparison with rats exposed to hypobaria under 21% O2. Exposure of rats to blast-induced acceleration of 100G increases cerebral axonal injury, which is significantly exacerbated by exposure to hypobaria as early as 6 hours and as late as 6 days postblast. Rats exposed to underbody blasts and then to hypobaria under 100% O2 exhibit increased axonal damage and impaired motor function compared to those subjected to blast and hypobaria under 21% O2. These findings raise concern about the effects of AE-related hypobaria on TBI victims, the timing of AE after TBI, and whether these effects

  5. FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle

    DEFF Research Database (Denmark)

    Lassiter, David G; Nylén, Carolina; Sjögren, Rasmus J O


    and the direct role of FAK on glucose and lipid metabolism. We hypothesised that insulin treatment and AMPK activation would have opposing effects on FAK phosphorylation and that gene silencing of FAK would alter metabolism. METHODS: Human muscle was treated with insulin or the AMPK-activating compound 5......-aminoimadazole-4-carboxamide ribonucleotide (AICAR) to determine FAK phosphorylation and glucose transport. Primary human skeletal muscle cells were used to study the effects of insulin or AICAR treatment on FAK signalling during serum starvation, as well as to determine the metabolic consequences of silencing...... in various non-muscle cell types and plays a regulatory role during skeletal muscle differentiation. The role of FAK in skeletal muscle in relation to insulin stimulation or AMPK activation is unknown. We examined the effects of insulin or AMPK activation on FAK phosphorylation in human skeletal muscle...

  6. Segregation of Axial Motor and Sensory Pathways via Heterotypic Trans-Axonal Signaling (United States)

    Gallarda, Benjamin W.; Bonanomi, Dario; Müller, Daniel; Brown, Arthur; Alaynick, William A.; Andrews, Shane E.; Lemke, Greg; Pfaff, Samuel L.; Marquardt, Till


    Execution of motor behaviors relies on circuitries effectively integrating immediate sensory feedback to efferent pathways controlling muscle activity. It remains unclear how, during neuromuscular circuit assembly, sensory and motor projections become incorporated into tightly coordinated, yet functionally separate pathways. We report that, within axial nerves, establishment of discrete afferent and efferent pathways depends on coordinate signaling between coextending sensory and motor projections. These heterotypic axon-axon interactions require motor axonal EphA3/EphA4 receptor tyrosine kinases activated by cognate sensory axonal ephrin-A ligands. Genetic elimination of trans-axonal ephrin-A → EphA signaling in mice triggers drastic motor-sensory miswiring, culminating in functional efferents within proximal afferent pathways. Effective assembly of a key circuit underlying motor behaviors thus critically depends on trans-axonal signaling interactions resolving motor and sensory projections into discrete pathways. PMID:18403711

  7. A Combinatorial Approach to Induce Sensory Axon Regeneration into the Dorsal Root Avulsed Spinal Cord

    DEFF Research Database (Denmark)

    Hoeber, Jan; Konig, Niclas; Trolle, Carl


    restores sensory functions. In this study, we elucidate mechanisms underlying stem cell-mediated ingrowth of sensory axons after dorsal root avulsion (DRA). We show that human spinal cord neural stem/progenitor cells (hscNSPC), and also, mesoporous silica particles loaded with growth factor mimetics (Meso......MIM), supported sensory axon regeneration. However, when hscNSPC and MesoMIM were combined, sensory axon regeneration failed. Morphological and tracing analysis showed that sensory axons grow through the newly established glial scar along “bridges” formed by migrating stem cells. Coimplantation of Meso......MIM prevented stem cell migration, “bridges” were not formed, and sensory axons failed to enter the spinal cord. MesoMIM applied alone supported sensory axons ingrowth, but without affecting glial scar formation. In vitro, the presence of MesoMIM significantly impaired migration of hscNSPC without affecting...

  8. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.


    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...

  9. Inter-axonal interaction defines tiled presynaptic innervation in C. elegans


    Mizumoto, Kota; Shen, Kang


    Cellular interactions between neighboring axons are essential for global topographic map formation. Here we show that axonal interactions also precisely instruct the location of synapses. Motoneurons form en passant synapses in Caenorhabditis elegans. While axons from the same neuron class significantly overlap, each neuron innervates a unique and tiled segment of the muscle field by restricting its synapses to a distinct subaxonal domain—a phenomenon we term “synaptic tiling”. Using DA8 and ...

  10. Oligodendroglial MCT1 and Metabolic Support of Axons in Multiple Sclerosis (United States)


    AWARD NUMBER: W81XWH-14-1-0524 TITLE:Oligodendroglial MCT1 and Metabolic Support of Axons in Multiple Sclerosis PRINCIPAL INVESTIGATOR: Jeffrey D...29 Sep 2015 4. TITLE AND SUBTITLE Oligodendroglial MCT1 and Metabolic Support of Axons in Multiple Sclerosis 5a. CONTRACT NUMBER W81XWH-14-1-0524...MCT1 in injured oligodendroglia of multiple sclerosis patients contributes to axon neurodegeneration and that increasing MCT1 will be protective in the

  11. Acutely damaged axons are remyelinated in multiple sclerosis and experimental models of demyelination. (United States)

    Schultz, Verena; van der Meer, Franziska; Wrzos, Claudia; Scheidt, Uta; Bahn, Erik; Stadelmann, Christine; Brück, Wolfgang; Junker, Andreas


    Remyelination is in the center of new therapies for the treatment of multiple sclerosis to resolve and improve disease symptoms and protect axons from further damage. Although remyelination is considered beneficial in the long term, it is not known, whether this is also the case early in lesion formation. Additionally, the precise timing of acute axonal damage and remyelination has not been assessed so far. To shed light onto the interrelation between axons and the myelin sheath during de- and remyelination, we employed cuprizone- and focal lysolecithin-induced demyelination and performed time course experiments assessing the evolution of early and late stage remyelination and axonal damage. We observed damaged axons with signs of remyelination after cuprizone diet cessation and lysolecithin injection. Similar observations were made in early multiple sclerosis lesions. To assess the correlation of remyelination and axonal damage in multiple sclerosis lesions, we took advantage of a cohort of patients with early and late stage remyelinated lesions and assessed the number of APP- and SMI32- positive damaged axons and the density of SMI31-positive and silver impregnated preserved axons. Early de- and remyelinating lesions did not differ with respect to axonal density and axonal damage, but we observed a lower axonal density in late stage demyelinated multiple sclerosis lesions than in remyelinated multiple sclerosis lesions. Our findings suggest that remyelination may not only be protective over a long period of time, but may play an important role in the immediate axonal recuperation after a demyelinating insult. © 2017 The Authors GLIA Published by Wiley Periodicals, Inc.

  12. Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane. (United States)

    Zhang, Liang; Trushin, Sergey; Christensen, Trace A; Tripathi, Utkarsh; Hong, Courtney; Geroux, Rachel E; Howell, Kyle G; Poduslo, Joseph F; Trushina, Eugenia


    Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype. Copyright © 2018 The Authors. Published by

  13. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    Energy Technology Data Exchange (ETDEWEB)

    Karen S. Browning


    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  14. Ribosomes and polyribosomes are present in the squid giant axon: an immunocytochemical study. (United States)

    Sotelo, J R; Kun, A; Benech, J C; Giuditta, A; Morillas, J; Benech, C R


    Ribosomes and polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.

  15. Regulation of Axonal Midline Guidance by Prolyl 4-Hydroxylation in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Torpe, Nanna; Pocock, Roger David John


    , little is known of its importance in the control of axon guidance. In a screen of prolyl 4-hydroxylase (P4H) mutants, we found that genetic removal of a specific P4H subunit, DPY-18, causes dramatic defects in C. elegans neuroanatomy. In dpy-18 mutant animals, the axons of specific ventral nerve cord......Neuronal wiring during development requires that the growth cones of axons and dendrites are correctly guided to their appropriate targets. As in other animals, axon growth cones in Caenorhabditis elegans integrate information in their extracellular environment via interactions among transiently...

  16. Forced notch signaling inhibits commissural axon outgrowth in the developing chick central nerve system.

    Directory of Open Access Journals (Sweden)

    Ming Shi

    Full Text Available BACKGROUND: A collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the neural tubes of HH10-11 chick embryos were in ovo electroporated with various Notch transgenes of activating or inhibiting Notch signaling, and then their effects on commissural axon outgrowth across the floor plate midline in the chick developing central nerve system were investigated. Our results showed that forced expression of Notch intracellular domain, constitutively active form of RBPJ, or full-length Hes1 in the rostral hindbrain, diencephalon and spinal cord at stage HH10-11 significantly inhibited commissural axon outgrowth. On the other hand, inhibition of Notch signaling by ectopically expressing a dominant-negative form of RBPJ promoted commissural axonal growth along the circumferential axis. Further results revealed that these Notch signaling-mediated axon outgrowth defects may be not due to the alteration of axon guidance since commissural axon marker TAG1 was present in the axons in floor plate midline, and also not result from the changes in cell fate determination of commissural neurons since the expression of postmitotic neuron marker Tuj1 and specific commissural markers TAG1 and Pax7 was unchanged. CONCLUSIONS/SIGNIFICANCE: We first used an in vivo system to provide evidence that forced Notch signaling negatively regulates commissural axon outgrowth.

  17. Alterations in the Local Axonal Environment Influence Target Reinnervation and Neuronal Survival after Postnatal Axotomy

    National Research Council Canada - National Science Library

    Dainer, Hugh M


    Following peripheral nerve injury in adult animals, Schwann cells (SC) proliferate and provide guidance in the local axonal environment by generating the infrastructure along which regenerating nerves grow...

  18. N-Propionylmannosamine stimulates axonal elongation in a murine model of sciatic nerve injury

    Directory of Open Access Journals (Sweden)

    Christian Witzel


    Full Text Available Increasing evidence indicates that sialic acid plays an important role during nerve regeneration. Sialic acids can be modified in vitro as well as in vivo using metabolic oligosaccharide engineering of the N-acyl side chain. N-Propionylmannosamine (ManNProp increases neurite outgrowth and accelerates the reestablishment of functional synapses in vitro. We investigated the influence of systemic ManNProp application using a specific in vivo mouse model. Using mice expressing axonal fluorescent proteins, we quantified the extension of regenerating axons, the number of regenerating axons, the number of arborising axons and the number of branches per axon 5 days after injury. Sciatic nerves from non-expressing mice were grafted into those expressing yellow fluorescent protein. We began a twice-daily intraperitoneal application of either peracetylated ManNProp (200 mg/kg or saline solution 5 days before injury, and continued it until nerve harvest (5 days after transection. ManNProp significantly increased the mean distance of axonal regeneration (2.49 mm vs. 1.53 mm; P < 0.005 and the number of arborizing axons (21% vs. 16% P = 0.008 5 days after sciatic nerve grafting. ManNProp did not affect the number of regenerating axons or the number of branches per arborizing axon. The biochemical glycoengineering of the N-acyl side chain of sialic acid might be a promising approach for improving peripheral nerve regeneration.

  19. Segregation of ipsilateral retinal ganglion cell axons at the optic chiasm requires the Shh receptor Boc. (United States)

    Fabre, Pierre J; Shimogori, Tomomi; Charron, Frédéric


    The pattern of contralaterally and ipsilaterally projecting retinal ganglion cell (RGC) axons at the optic chiasm is essential for the establishment of binocular vision. Contralateral axons cross the chiasm midline as they progress from the optic nerve to the optic tract. In contrast, ipsilateral axons deviate from the chiasm and continue in the ipsilateral optic tract, avoiding the chiasm midline. The molecular mechanism underlying this phenomenon is not completely understood. Here we show that the Sonic Hedgehog (Shh) receptor Boc is enriched in ipsilateral RGCs of the developing retina. Together with the presence of Shh at the midline, this complementary expression pattern led us to hypothesize that Shh might repel ipsilateral RGC axons at the chiasm. Consistent with this hypothesis, we found that only Boc-positive RGC axons retract in vitro in response to Shh and that this response is lost in Boc mutant RGCs. In vivo, we show that Boc is required for the normal segregation of ipsilateral axons at the optic chiasm and, conversely, that Boc expression in contralateral RGCs prevents their axons from crossing the optic chiasm. Together, these results suggest that Shh repels ipsilateral RGC axons at the optic chiasm via its receptor Boc. This work identifies a novel molecular pathway required for the segregation of axons at the optic chiasm.

  20. Biomarker evidence of axonal injury in neuroasymptomatic HIV-1 patients.

    Directory of Open Access Journals (Sweden)

    Jan Jessen Krut

    Full Text Available Prevalence of neurocognitive impairment in HIV-1 infected patients is reported to be high. Whether this is a result of active HIV-related neurodegeneration is unclear. We examined axonal injury in HIV-1 patients by measuring the light subunit of neurofilament protein (NFL in CSF with a novel, sensitive method.With a cross-sectional design, CSF concentrations of neurofilament protein light (NFL (marker of neuronal injury, neopterin (intrathecal immunoactivation and CSF/Plasma albumin ratio (blood-brain barrier integrity were analyzed on CSF from 252 HIV-infected patients, subdivided into untreated neuroasymptomatics (n = 200, HIV-associated dementia (HAD (n = 14 and on combinations antiretroviral treatment (cART (n = 85, and healthy controls (n = 204. 46 HIV-infected patients were included in both treated and untreated groups, but sampled at different timepoints. Furthermore, 78 neuroasymptomatic patients were analyzed before and after treatment initiation.While HAD patients had the highest NFL concentrations, elevated CSF NFL was also found in 33% of untreated neuroasymptomatic patients, mainly in those with blood CD4+ cell counts below 250 cells/μL. CSF NFL concentrations in the untreated neuroasymptomatics and treated groups were equivalent to controls 18.5 and 3.9 years older, respectively. Neopterin correlated with NFL levels in untreated groups while the albumin ratio correlated with NFL in both untreated and treated groups.Increased CSF NFL indicates ongoing axonal injury in many neuroasymptomatic patients. Treatment decreases NFL, but treated patients retain higher levels than controls, indicating either continued virus-related injury or an aging-like effect of HIV infection. NFL correlates with neopterin and albumin ratio, suggesting an association between axonal injury, neuroinflammation and blood-brain barrier permeability. NFL appears to be a sensitive biomarker of subclinical and clinical brain injury in HIV and warrants further

  1. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    International Nuclear Information System (INIS)

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.


    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

  2. Mechanisms of hyperpolarization in regenerated mature motor axons in cat

    DEFF Research Database (Denmark)

    Moldovan, Mihai; Krarup, Christian


    We found persistent abnormalities in the recovery of membrane excitability in long-term regenerated motor nerve fibres in the cat as indicated in the companion paper. These abnormalities could partly be explained by membrane hyperpolarization. To further investigate this possibility, we compared...... the changes in excitability in control nerves and long-term regenerated cat nerves (3-5 years after tibial nerve crush) during manoeuvres known to alter axonal membrane Na(+)-K(+) pump function: polarization, cooling to 20 degrees C, reperfusion after 10 min ischaemia, and up to 60 s of repetitive stimulation...

  3. Mitochondrial oxidative phosphorylation in autosomal dominant optic atrophy

    Directory of Open Access Journals (Sweden)

    Cline Susan D


    Full Text Available Abstract Background Autosomal dominant optic atrophy (ADOA, a form of progressive bilateral blindness due to loss of retinal ganglion cells and optic nerve deterioration, arises predominantly from mutations in the nuclear gene for the mitochondrial GTPase, OPA1. OPA1 localizes to mitochondrial cristae in the inner membrane where electron transport chain complexes are enriched. While OPA1 has been characterized for its role in mitochondrial cristae structure and organelle fusion, possible effects of OPA1 on mitochondrial function have not been determined. Results Mitochondria from six ADOA patients bearing OPA1 mutations and ten ADOA patients with unidentified gene mutations were studied for respiratory capacity and electron transport complex function. Results suggest that the nuclear DNA mutations that give rise to ADOA in our patient population do not alter mitochondrial electron transport. Conclusion We conclude that the pathophysiology of ADOA likely stems from the role of OPA1 in mitochondrial structure or fusion and not from OPA1 support of oxidative phosphorylation.

  4. Bruchpilot in ribbon-like axonal agglomerates, behavioral defects, and early death in SRPK79D kinase mutants of Drosophila.

    Directory of Open Access Journals (Sweden)

    Vanessa Nieratschker


    Full Text Available Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP protein is associated with T-shaped ribbons ("T-bars" at presynaptic active zones (AZs. BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, we have identified a P-transposon insertion in gene CG11489 (location 79D which shows high homology to mammalian genes for SR protein kinases (SRPKs. SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins. Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, we name CG11489 the Drosophila Srpk79D gene. We have characterized Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. We propose that the Drosophila Srpk79D gene characterized here may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the

  5. Glial scar and axonal regeneration in the CNS: lessons from GFAP and vimentin transgenic mice. (United States)

    Ribotta, M G; Menet, V; Privat, A


    Astrocytes play an active role in the brain and spinal cord. For example, they have a function in formation and maintenance of the blood-brain barrier, ion homeostasis, neurotransmitter transport, production of extracellular matrix, and neuromodulation. Moreover, they play a role in preserving or even restoring the structural and physiological integrity after tissue injury. Currently, the function of astrocytes was studied with regard to the controversially discussed aspects of permissivity on the one-hand-side and inhibition of the other side exerted by reactive astrocytes for axonal regrowth in the adult CNS. Accordingly, knock-out mice deficient in vimentin (VIM) and/or glial fibrillary acidic protein (GFAP), the two major IF-proteins of astrocytes, were investigated. In addition, in vitro studies were carried out, on whether the absence of one or both proteins (VIM, GFAP) influences axonal regeneration. In experimental animals, a hemisection of the spinal cord was performed utilizing the above mentioned double-mutant mice. The knock-out mice were generated by gene targeting. Double-mutants were obtained by crossing single null mice. The in vitro results indicate that both VIM and GFAP were absent in astrocytic cultures obtained from double-mutant mice. On the other side, the proteins were detected in more than 85%, of cultured cells from wild types. Co-culture of mutant mice astrocytes with neurons revealed that the neuronal density was different from that obtained in culture with wild type astrocytes. On the other side, there was a marked increase in neuronal density in co-cultures utilizing both GFAP knock-out- or double-mutant mice astrocytes again as compared to co-cultures with wild type astrocytes. Moreover, the neurite length of neurons was significantly increased in experiments with neurons growing on astrocytes from GFAP-knock-out or double-mutant mice. The in vivo experiments demonstrate an increase of nestin (NES) immunoreactivity at three days in

  6. Gogo receptor contributes to retinotopic map formation and prevents R1-6 photoreceptor axon bundling.

    Directory of Open Access Journals (Sweden)

    Irina Hein

    Full Text Available BACKGROUND: Topographic maps form the basis of neural processing in sensory systems of both vertebrate and invertebrate species. In the Drosophila visual system, neighboring R1-R6 photoreceptor axons innervate adjacent positions in the first optic ganglion, the lamina, and thereby represent visual space as a continuous map in the brain. The mechanisms responsible for the establishment of retinotopic maps remain incompletely understood. RESULTS: Here, we show that the receptor Golden goal (Gogo is required for R axon lamina targeting and cartridge elongation in a partially redundant fashion with local guidance cues provided by neighboring axons. Loss of function of Gogo in large clones of R axons results in aberrant R1-R6 fascicle spacing. Gogo affects target cartridge selection only indirectly as a consequence of the disordered lamina map. Interestingly, small clones of gogo deficient R axons perfectly integrate into a proper retinotopic map suggesting that surrounding R axons of the same or neighboring fascicles provide complementary spatial guidance. Using single photoreceptor type rescue, we show that Gogo expression exclusively in R8 cells is sufficient to mediate targeting of all photoreceptor types in the lamina. Upon lamina targeting and cartridge selection, R axons elongate within their individual cartridges. Interestingly, here Gogo prevents bundling of extending R1-6 axons. CONCLUSION: Taken together, we propose that Gogo contributes to retinotopic map formation in the Drosophila lamina by controlling the distribution of R1-R6 axon fascicles. In a later developmental step, the regular position of R1-R6 axons along the lamina plexus is crucial for target cartridge selection. During cartridge elongation, Gogo allows R1-R6 axons to extend centrally in the lamina cartridge.

  7. N-cadherin regulates primary motor axon growth and branching during zebrafish embryonic development. (United States)

    Brusés, Juan L


    N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type-specific pathway selection. Analysis of N-cadherin mutants (cdh2(hi3644Tg) ) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ∼40% of the somitic hemisegments and an ∼150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point that abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to stall abnormally at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin-depleted embryos, the majority of primary motor axons innervated their appropriate myotomal territories, indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection. Copyright © 2011 Wiley-Liss, Inc.

  8. Plexin A3 and turnout regulate motor axonal branch morphogenesis in zebrafish.

    Directory of Open Access Journals (Sweden)

    Rajiv Sainath

    Full Text Available During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch

  9. Flux control through protein phosphorylation in yeast

    DEFF Research Database (Denmark)

    Chen, Yu; Nielsen, Jens


    Protein phosphorylation is one of the most important mechanisms regulating metabolism as it can directly modify metabolic enzymes by the addition of phosphate groups. Attributed to such a rapid and reversible mechanism, cells can adjust metabolism rapidly in response to temporal changes. The yeast...... as well as identify mechanisms underlying human metabolic diseases. Here we collect functional phosphorylation events of 41 enzymes involved in yeast metabolism and demonstrate functional mechanisms and the application of this information in metabolic engineering. From a systems biology perspective, we...... describe the development of phosphoproteomics in yeast as well as approaches to analysing the phosphoproteomics data. Finally, we focus on integrated analyses with other omics data sets and genome-scale metabolic models. Despite the advances, future studies improving both experimental technologies...

  10. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. (United States)

    Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Gingras, Marie-Claude; Muthuswamy, Lakshmi B; Johns, Amber L; Miller, David K; Wilson, Peter J; Patch, Ann-Marie; Wu, Jianmin; Chang, David K; Cowley, Mark J; Gardiner, Brooke B; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J; Gill, Anthony J; Pinho, Andreia V; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R Scott; Humphris, Jeremy L; Kaplan, Warren; Jones, Marc D; Colvin, Emily K; Nagrial, Adnan M; Humphrey, Emily S; Chou, Angela; Chin, Venessa T; Chantrill, Lorraine A; Mawson, Amanda; Samra, Jaswinder S; Kench, James G; Lovell, Jessica A; Daly, Roger J; Merrett, Neil D; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M; Fisher, William E; Brunicardi, F Charles; Hodges, Sally E; Reid, Jeffrey G; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R; Dinh, Huyen; Buhay, Christian J; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E; Yung, Christina K; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A; Petersen, Gloria M; Gallinger, Steven; Hruban, Ralph H; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Schulick, Richard D; Wolfgang, Christopher L; Morgan, Richard A; Lawlor, Rita T; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A; Mann, Karen M; Jenkins, Nancy A; Perez-Mancera, Pedro A; Adams, David J; Largaespada, David A; Wessels, Lodewyk F A; Rust, Alistair G; Stein, Lincoln D; Tuveson, David A; Copeland, Neal G; Musgrove, Elizabeth A; Scarpa, Aldo; Eshleman, James R; Hudson, Thomas J; Sutherland, Robert L; Wheeler, David A; Pearson, John V; McPherson, John D; Gibbs, Richard A; Grimmond, Sean M


    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

  11. Imaging findings in diffuse axonal injury after closed head trauma

    Energy Technology Data Exchange (ETDEWEB)

    Parizel, P.M.; Oezsarlak, Oe.; Goethem, J.W. van; Hauwe, L. van den; Schepper, A.M. de [Department of Radiology, Universitair Ziekenhuis Antwerpen (University of Antwerp), Edegem (Belgium); Dillen, C.; Cosyns, P. [Department of Psychiatry, Universitair Ziekenhuis Antwerpen (University of Antwerp), Edegem (Belgium); Verlooy, J. [Department of Neurosurgery, Universitair Ziekenhuis Antwerpen (University of Antwerp), Edegem (Belgium)


    Even in patients with closed head trauma, brain parenchyma can be severely injured due to disruption of axonal fibers by shearing forces during acceleration, deceleration, and rotation of the head. In this article we review the spectrum of imaging findings in patients with diffuse axonal injuries (DAI) after closed head trauma. Knowledge of the location and imaging characteristics of DAI is important to radiologists for detection and diagnosis. Common locations of DAI include: cerebral hemispheric gray-white matter interface and subcortical white matter, body and splenium of corpus callosum, basal ganglia, dorsolateral aspect of brainstem, and cerebellum. In the acute phase, CT may show punctate hemorrhages. The true extent of brain involvement is better appreciated with MR imaging, because both hemorrhagic and non-hemorrhagic lesions (gliotic scars) can be detected. The MR appearance of DAI lesions depends on several factors, including age of injury, presence of hemorrhage or blood-breakdown products (e. g., hemosiderin), and type of sequence used. Technical aspects in MR imaging of these patients are discussed. Non-hemorrhagic lesions can be detected with fluid attenuated inversion recovery (FLAIR), proton-density-, or T2-weighted images, whereas gradient echo sequences with long TE increase the visibility of old hemorrhagic lesions. (orig.) With 12 figs., 12 refs.

  12. Rapid signaling in distinct dopaminergic axons during locomotion and reward (United States)

    Howe, MW; Dombeck, DA


    Summary Dopaminergic projections from the midbrain to striatum are critical for motor control, as their degeneration in Parkinson’s disease results in profound movement deficits. Paradoxically, most recording methods report rapid phasic dopamine signaling (~100ms bursts) to unpredicted rewards, with little evidence for movement-related signaling. The leading model posits that phasic signaling in striatum targeting dopamine neurons drive reward-based learning, while slow variations in firing (tens of seconds to minutes) in these same neurons bias animals towards or away from movement. However, despite widespread acceptance of this model, current methods have provided little evidence to support or refute it. Here, using new optical recording methods, we report the discovery of rapid phasic signaling in striatum-targeting dopaminergic axons that was associated with, and capable of triggering, locomotion in mice. Axons expressing these signals were largely distinct from those signaling during unexpected rewards. These results suggest that dopaminergic neuromodulation can differentially impact motor control and reward learning with sub-second precision and suggest that both precise signal timing and neuronal subtype are important parameters to consider in the treatment of dopamine-related disorders. PMID:27398617

  13. Targeting PCNA Phosphorylation in Breast Cancer (United States)


    inhibitors at high concentrations . Detection of PCNA Phosphorylation upon Kinase Inhibitor Treatment in MDA-MB-468 Breast Cancer Cells A panel of...Bradford assay to determine protein concentration . Bradford Assay and Total Protein Levels amongst Kinase Treatments The Bradford assay to determine...washed with water (2 x), Sat. aq. NaHCO3 (1 x) and brine . After being dried over Na2SO4, the solvent was concentrated under vacuum and the residue was

  14. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan


    In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

  15. Synthesis and characterization of -phosphorylated thioureas ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 122; Issue 3. Synthesis and characterization of -phosphorylated thioureas RNHC(S)NHP(O)(OPr)2 (R = 2-MeC6H4, 2,6-Me2C6H3, 2,4,6-Me3C6H2). Damir A Safin Maria G Babashkina Michael Bolte Axel Klein. Full Papers Volume 122 Issue 3 May 2010 pp 409- ...

  16. Regulation of NMDA Receptors by Phosphorylation


    Chen, Bo-Shiun; Roche, Katherine W.


    N-methyl-D-aspartate (NMDA) receptors are critical for neuronal development and synaptic plasticity. The molecular mechanisms underlying the synaptic localization and functional regulation of NMDA receptors have been the subject of extensive studies. In particular, phosphorylation has emerged as a fundamental mechanism that regulates NMDA receptor trafficking and can alter the channel properties of NMDA receptors. Here we summarize recent advances in the characterization of NMDA receptor phos...

  17. Systematic inference of functional phosphorylation events in yeast metabolism

    DEFF Research Database (Denmark)

    Chen, Yu; Wang, Yonghong; Nielsen, Jens


    of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality...... biology....

  18. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Brunak, Søren; Olsen, JV


    ) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells....

  19. Phosphorylation Stoichiometries of Human Eukaryotic Initiation Factors

    Directory of Open Access Journals (Sweden)

    Armann Andaya


    Full Text Available Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors, this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.

  20. Mixed mechanisms of multi-site phosphorylation. (United States)

    Suwanmajo, Thapanar; Krishnan, J


    Multi-site phosphorylation is ubiquitous in cell biology and has been widely studied experimentally and theoretically. The underlying chemical modification mechanisms are typically assumed to be distributive or processive. In this paper, we study the behaviour of mixed mechanisms that can arise either because phosphorylation and dephosphorylation involve different mechanisms or because phosphorylation and/or dephosphorylation can occur through a combination of mechanisms. We examine a hierarchy of models to assess chemical information processing through different mixed mechanisms, using simulations, bifurcation analysis and analytical work. We demonstrate how mixed mechanisms can show important and unintuitive differences from pure distributive and processive mechanisms, in some cases resulting in monostable behaviour with simple dose-response behaviour, while in other cases generating new behaviour-like oscillations. Our results also suggest patterns of information processing that are relevant as the number of modification sites increases. Overall, our work creates a framework to examine information processing arising from complexities of multi-site modification mechanisms and their impact on signal transduction. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  1. Phosphorylation of erythrocyte membrane liberates calcium

    International Nuclear Information System (INIS)

    Chauhan, V.P.S.; Brockerhoff, H.


    Phosphorylation of permeabilized erythrocyte ghost membranes with ATP results in an increase free calcium level as measured with the help of Ca 2+ electrode and 45 Ca. This effect could not be observed in the presence of p - chloromercuric benzoate, an inhibitor of kinases. The rise in the free calcium due to phosphorylation of the membrane was accompanied by a decrease in the level of phosphatidylinositol (PI) and an increase in phosphatidylinositolmonophosphate (PIP) and phosphatidylinositolbisphosphate (PIP 2 ). These results support the proposal that an inositol shuttle, PI ↔ PIP ↔ PIP 2 , operates to maintain the intracellular calcium concentration. The cation is believed to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with the participation of both PO and fatty acid ester CO groups. When the inositol group of such a cage is phosphorylated, inter-headgroup hydrogen bonding between the lipids is broken. As a result the cage opens and calcium is released

  2. The role of mitochondria in axonal degeneration and tissue repair in MS

    NARCIS (Netherlands)

    van Horssen, J.; Witte, M.E.; Ciccarelli, O.


    Axonal injury is a key feature of multiple sclerosis (MS) pathology and is currently seen as the main correlate for permanent clinical disability. Although little is known about the pathogenetic mechanisms that drive axonal damage and loss, there is accumulating evidence highlighting the central

  3. Structure and Function of an Actin-Based Filter in the Proximal Axon

    Directory of Open Access Journals (Sweden)

    Varuzhan Balasanyan


    Full Text Available Summary: The essential organization of microtubules within neurons has been described; however, less is known about how neuronal actin is arranged and the functional implications of its arrangement. Here, we describe, in live cells, an actin-based structure in the proximal axon that selectively prevents some proteins from entering the axon while allowing the passage of others. Concentrated patches of actin in proximal axons are present shortly after axonal specification in rat and zebrafish neurons imaged live, and they mark positions where anterogradely traveling vesicles carrying dendritic proteins halt and reverse. Patches colocalize with the ARP2/3 complex, and when ARP2/3-mediated nucleation is blocked, a dendritic protein mislocalizes to the axon. Patches are highly dynamic, with few persisting longer than 30 min. In neurons in culture and in vivo, actin appears to form a contiguous, semipermeable barrier, despite its apparently sparse distribution, preventing axonal localization of constitutively active myosin Va but not myosin VI. : Balasanyan et al. find dynamic patches of actin in proximal axons of live neurons, mature and newly differentiated, in culture and in vivo. Patches contribute to a filter that sequesters some proteins within the somatodendritic domain while allowing others to pass into the axon, leading to polarized localization of proteins.

  4. The progeroid gene BubR1 regulates axon myelination and motor function

    NARCIS (Netherlands)

    Choi, C.I.; Yoo, K.H.; Hussaini, S.M.; Jeon, B.T.; Welby, J.; Gan, H.; Scarisbrick, I.A.; Zhang, Z.; Baker, D.J.; Deursen, J.M.A. van; Rodriguez, M.; Jang, M.H.


    Myelination, the process by which oligodendrocytes form the myelin sheath around axons, is key to axonal signal transduction and related motor function in the central nervous system (CNS). Aging is characterized by degenerative changes in the myelin sheath, although the molecular underpinnings of

  5. Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

    Directory of Open Access Journals (Sweden)

    Yihao Zhang


    Full Text Available Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav, which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

  6. Axonal and presynaptic protein synthesis: new insights into the biology of the neuron

    NARCIS (Netherlands)

    Giuditta, A.; Kaplan, B.B.; van Minnen, J.; Alvarez, J.; Koenig, E.


    The presence of a local mRNA translation system in axons and terminals was proposed almost 40 years ago. Over the ensuing period, an impressive body of evidence has grown to support this proposal - yet the nerve cell body is still considered to be the only source of axonal and presynaptic proteins.

  7. Blast overpressure induced axonal injury changes in rat brainstem and spinal cord

    Directory of Open Access Journals (Sweden)

    Srinivasu Kallakuri


    Full Text Available Introduction: Blast induced neurotrauma has been the signature wound in returning soldiers from the ongoing wars in Iraq and Afghanistan. Of importance is understanding the pathomechansim(s of blast overpressure (OP induced axonal injury. Although several recent animal models of blast injury indicate the neuronal and axonal injury in various brain regions, animal studies related to axonal injury in the white matter (WM tracts of cervical spinal cord are limited. Objective: The purpose of this study was to assess the extent of axonal injury in WM tracts of cervical spinal cord in male Sprague Dawley rats subjected to a single insult of blast OP. Materials and Methods: Sagittal brainstem sections and horizontal cervical spinal cord sections from blast and sham animals were stained by neurofilament light (NF-L chain and beta amyloid precursor protein immunocytochemistry and observed for axonal injury changes. Results: Observations from this preliminary study demonstrate axonal injury changes in the form of prominent swellings, retraction bulbs, and putative signs of membrane disruptions in the brainstem and cervical spinal cord WM tracts of rats subjected to blast OP. Conclusions: Prominent axonal injury changes following the blast OP exposure in brainstem and cervical spinal WM tracts underscores the need for careful evaluation of blast induced injury changes and associated symptoms. NF-L immunocytochemistry can be considered as an additional tool to assess the blast OP induced axonal injury.

  8. Schwann Cell and Axon: An Interlaced Unit-From Action Potential to Phenotype Expression. (United States)

    Court, Felipe A; Alvarez, Jaime


    Here we propose a model of a peripheral axon with a great deal of autonomy from its cell body-the autonomous axon-but with a substantial dependence on its ensheathing Schwann cell (SC), the axon-SC unit. We review evidence in several fields and show that (i) axons can extend sprouts and grow without the concurrence of the cell body, but regulated by SCs; (ii) axons synthesize their proteins assisted by SCs that supply them with ribosomes and, probably, with mRNAs by way of exosomes; (iii) the molecular organization of the axoplasm, i.e., its phenotype, is regulated by the SC, as illustrated by the axonal microtubular content, which is down-regulated by the SC; and (iv) the axon has a program for self-destruction that is boosted by the SC. The main novelty of this model axon-SC unit is that it breaks with the notion that all proteins of the nerve cell are specified by its own nucleus. The notion of a collaborative specification of the axoplasm by more than one nucleus, which we present here, opens a new dimension in the understanding of the nervous system in health and disease and is also a frame of reference to understand other tissues or cell associations.

  9. N-docosahexaenoylethanolamine regulates Hedgehog signaling and promotes growth of cortical axons

    Directory of Open Access Journals (Sweden)

    Giorgi Kharebava


    Full Text Available Axonogenesis, a process for the establishment of neuron connectivity, is central to brain function. The role of metabolites derived from docosahexaenoic acid (DHA, 22:6n-3 that is specifically enriched in the brain, has not been addressed in axon development. In this study, we tested if synaptamide (N-docosahexaenoylethanolamine, an endogenous metabolite of DHA, affects axon growth in cultured cortical neurons. We found that synaptamide increased the average axon length, inhibited GLI family zinc finger 1 (GLI1 transcription and sonic hedgehog (Shh target gene expression while inducing cAMP elevation. Similar effects were produced by cyclopamine, a regulator of the Shh pathway. Conversely, Shh antagonized elevation of cAMP and blocked synaptamide-mediated increase in axon length. Activation of Shh pathway by a smoothened (SMO agonist (SAG or overexpression of SMO did not inhibit axon growth mediated by synaptamide or cyclopamine. Instead, adenylate cyclase inhibitor SQ22536 abolished synaptamide-mediated axon growth indicating requirement of cAMP elevation for this process. Our findings establish that synaptamide promotes axon growth while Shh antagonizes synaptamide-mediated cAMP elevation and axon growth by a SMO-independent, non-canonical pathway.

  10. Misdirection and guidance of regenerating axons after experimental nerve injury and repair

    NARCIS (Netherlands)

    de Ruiter, Godard C W; Spinner, Robert J; Verhaagen, J.; Malessy, Martijn J A

    Misdirection of regenerating axons is one of the factors that can explain the limited results often found after nerve injury and repair. In the repair of mixed nerves innervating different distal targets (skin and muscle), misdirection may, for example, lead to motor axons projecting toward skin,

  11. Misdirection and guidance of regenerating axons after experimental nerve injury and repair A review

    NARCIS (Netherlands)

    Ruiter, G.C.W.; Spinner, R.J.; Verhaagen, J.; Malessay, M.J.A.


    Misdirection of regenerating axons is one of the factors that can explain the limited results often found after nerve injury and repair. In the repair of mixed nerves innervating different distal targets (skin and muscle), misdirection may, for example, lead to motor axons projecting toward skin,

  12. Misdirection and guidance of regenerating motor axons after experimental nerve injury and repair

    NARCIS (Netherlands)

    Ruiter, Godard de


    Misdirection of regenerating motor axons is one of the factors that can explain the disappointing recovery of function often observed after nerve injury and repair. In the first part of this thesis we quantified misdirection of motor axon regeneration after different types of nerve injury and repair

  13. Frizzled3 controls axonal polarity and intermediate target entry during striatal pathway development

    NARCIS (Netherlands)

    Morello, Francesca; Prasad, Asheeta A.; Rehberg, Kati; Baptista Vieira de Sá, Renata; Antón-Bolaños, Noelia; Leyva-Diaz, Eduardo; Adolfs, Youri; Tissir, Fadel; López-Bendito, Guillermina; Pasterkamp, R. Jeroen


    The striatum is a large brain nucleus with an important role in the control of movement and emotions.Mediumspiny neurons (MSNs) are striatal output neurons forming prominent descending axon tracts that target different brain nuclei. However, how MSN axon tracts in the forebrain develop remains

  14. Axon-somatic back-propagation in detailed models of spinal alpha motoneurons

    Directory of Open Access Journals (Sweden)

    Pietro eBalbi


    Full Text Available Antidromic action potentials following distal stimulation of motor axons occasionally fail to invade the soma of alpha motoneurons in spinal cord, due to their passing through regions of high non-uniformity.Morphologically detailed conductance-based models of cat spinal alpha motoneurons have been developed, with the aim to reproduce and clarify some aspects of the electrophysiological behavior of the antidromic axon-somatic spike propagation. Fourteen 3D morphologically detailed somata and dendrites of cat spinal alpha motoneurons have been imported from an open-access web-based database of neuronal morphologies,, and instantiated in neurocomputational models. An axon hillock, an axonal initial segment and a myelinated axon are added to each model.By sweeping the diameter of the axonal initial segment (AIS and the axon hillock, as well as the maximal conductances of sodium channels at the AIS and at the soma, the developed models are able to show the relationships between different geometric and electrophysiological configurations and the voltage attenuation of the antidromically travelling wave.In particular, a greater than usually admitted sodium conductance at AIS is necessary and sufficient to overcome the dramatic voltage attenuation occurring during antidromic spike propagation both at the myelinated axon-AIS and at the AIS-soma transitions.

  15. Axonal remodeling in the corticospinal tract after stroke: how does rehabilitative training modulate it?

    Directory of Open Access Journals (Sweden)

    Naohiko Okabe


    Full Text Available Stroke causes long-term disability, and rehabilitative training is commonly used to improve the consecutive functional recovery. Following brain damage, surviving neurons undergo morphological alterations to reconstruct the remaining neural network. In the motor system, such neural network remodeling is observed as a motor map reorganization. Because of its significant correlation with functional recovery, motor map reorganization has been regarded as a key phenomenon for functional recovery after stroke. Although the mechanism underlying motor map reorganization remains unclear, increasing evidence has shown a critical role for axonal remodeling in the corticospinal tract. In this study, we review previous studies investigating axonal remodeling in the corticospinal tract after stroke and discuss which mechanisms may underlie the stimulatory effect of rehabilitative training. Axonal remodeling in the corticospinal tract can be classified into three types based on the location and the original targets of corticospinal neurons, and it seems that all the surviving corticospinal neurons in both ipsilesional and contralesional hemisphere can participate in axonal remodeling and motor map reorganization. Through axonal remodeling, corticospinal neurons alter their output selectivity from a single to multiple areas to compensate for the lost function. The remodeling of the corticospinal axon is influenced by the extent of tissue destruction and promoted by various therapeutic interventions, including rehabilitative training. Although the precise molecular mechanism underlying rehabilitation-promoted axonal remodeling remains elusive, previous data suggest that rehabilitative training promotes axonal remodeling by upregulating growth-promoting and downregulating growth-inhibiting signals.

  16. Sensory axon-derived neuregulin-1 is required for axoglial signaling and normal sensory function but not for long-term axon maintenance

    DEFF Research Database (Denmark)

    Fricker, F.R.; Zhu, N.; Tsantoulas, C.


    Neuregulin-1 has a key role in mediating signaling between axons and Schwann cells during development. A limitation to studying its role in adulthood is the embryonic lethality of global Nrg1 gene deletion. We used the Cre-loxP system to generate transgenic mice in which neuregulin-1 is condition......Neuregulin-1 has a key role in mediating signaling between axons and Schwann cells during development. A limitation to studying its role in adulthood is the embryonic lethality of global Nrg1 gene deletion. We used the Cre-loxP system to generate transgenic mice in which neuregulin-1...... is conditionally ablated in the majority of small-diameter and a proportion of large-diameter sensory neurons that have axons conducting in the C- and Adelta-fiber range, respectively. Sensory neuron-specific neuregulin-1 ablation resulted in abnormally large Remak bundles with axons clustered in "polyaxonal...... cells required for normal sensory function. Sensory neuronal survival and axonal maintenance, however, are not dependent on axon-derived neuregulin-1 signaling in adulthood Udgivelsesdato: 2009/6/17...

  17. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    International Nuclear Information System (INIS)

    Smet-Nocca, Caroline; Launay, Hélène; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle


    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1 H, 15 N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  18. Intra-axonal Synthesis of SNAP25 Is Required for the Formation of Presynaptic Terminals

    Directory of Open Access Journals (Sweden)

    Andreia F.R. Batista


    Full Text Available Localized protein synthesis is a mechanism for developing axons to react acutely and in a spatially restricted manner to extracellular signals. As such, it is important for many aspects of axonal development, but its role in the formation of presynapses remains poorly understood. We found that the induced assembly of presynaptic terminals required local protein synthesis. Newly synthesized proteins were detectable at nascent presynapses within 15 min of inducing synapse formation in isolated axons. The transcript for the t-SNARE protein SNAP25, which is required for the fusion of synaptic vesicles with the plasma membrane, was recruited to presynaptic sites and locally translated. Inhibition of intra-axonal SNAP25 synthesis affected the clustering of SNAP25 and other presynaptic proteins and interfered with the release of synaptic vesicles from presynaptic sites. This study reveals a critical role for the axonal synthesis of SNAP25 in the assembly of presynaptic terminals.

  19. JMJD-1.2/PHF8 controls axon guidance by regulating Hedgehog-like signaling

    DEFF Research Database (Denmark)

    Riveiro, Alba; Mariani, Luca; Malmberg, Kim Emily


    Components of the KDM7 family of histone demethylases are implicated in neuronal development and one member, PHF8, is often found to be mutated in cases of X-linked mental retardation. However, how PHF8 regulates neurodevelopmental processes and contributes to the disease is still largely unknown...... the axonal defects. Deficiency of either wrt-8 or grl-16, or reduced expression of homologs of genes promoting Hedgehog signaling, restores correct axon guidance in jmjd-1.2 mutants. Genetic and overexpression data indicate that Hedgehog-related genes act on axon guidance through actin remodelers. Thus, our...... study highlights a novel function of jmjd-1.2 in axon guidance that might be relevant for the onset of X-linked mental retardation and provides compelling evidence of a conserved function of the Hedgehog pathway in C. elegans axon migration....

  20. Lost in the jungle: new hurdles for optic nerve axon regeneration. (United States)

    Pernet, Vincent; Schwab, Martin E


    The poor regenerative capacity of injured central nervous system (CNS) axons leads to permanent neurological deficits after brain, spinal cord, or optic nerve lesions. In the optic nerve, recent studies showed that stimulation of the cytokine or mammalian target of rapamycin (mTOR) signaling pathways potently enhances sprouting and regeneration of injured retinal ganglion cell axons in adult mice, but does not allow the majority of axons to reach their main cerebral targets. New analyses have revealed axon navigation defects in the optic nerve and at the optic chiasm under conditions of strong growth stimulation. We propose that a balanced growth stimulatory treatment will have to be combined with guidance factors and suppression of local growth inhibitory factors to obtain the full regeneration of long CNS axonal tracts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay

    DEFF Research Database (Denmark)

    Colak, Dilek; Ji, Sheng-Jian; Porse, Bo T


    Growth cones enable axons to navigate toward their targets by responding to extracellular signaling molecules. Growth-cone responses are mediated in part by the local translation of axonal messenger RNAs (mRNAs). However, the mechanisms that regulate local translation are poorly understood. Here we...... show that Robo3.2, a receptor for the Slit family of guidance cues, is synthesized locally within axons of commissural neurons. Robo3.2 translation is induced by floor-plate-derived signals as axons cross the spinal cord midline. Robo3.2 is also a predicted target of the nonsense-mediated mRNA decay.......2 expression. These data show that local translation is regulated by mRNA stability and that NMD acts locally to influence axonal pathfinding....

  2. PACSIN1, a Tau-interacting protein, regulates axonal elongation and branching by facilitating microtubule instability. (United States)

    Liu, Yingying; Lv, Kaosheng; Li, Zenglong; Yu, Albert C H; Chen, Jianguo; Teng, Junlin


    Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity.

  3. Axonal sprouting regulates myelin basic protein gene expression in denervated mouse hippocampus

    DEFF Research Database (Denmark)

    Jensen, M B; Poulsen, F R; Finsen, B


    to 35 days after transection of the entorhino-hippocampal perforant path axonal projection. In situ hybridization analysis showed that anterograde axonal and terminal degeneration lead to upregulated oligodendrocyte MBP mRNA expression starting between day 2 and day 4, in (1) the deep part of stratum...... axonal and terminal degeneration, myelin degenerative changes, microglial activation and axotomi-induced axonal sprouting. Oligodendrocyte MBP mRNA expression reached maximum in both these areas at day 7. MBP gene transcription remained constant in stratum radiatum, stratum pyramidale and stratum oriens...... of CA1, areas that were unaffected by perforant path transection. These results provide strong evidence that oligodendrocyte MBP gene expression can be regulated by axonal sprouting independently of microglial activation in the injured adult CNS....

  4. Activity-dependent myelination of parvalbumin interneurons mediated by axonal morphological plasticity. (United States)

    Stedehouder, J; Brizee, D; Shpak, G; Kushner, S A


    Axonal myelination of neocortical pyramidal neurons is dynamically modulated by neuronal activity. Recent studies have shown that a substantial proportion of neocortical myelin content is contributed by fast-spiking, parvalbumin (PV)-positive interneurons. However, it remains unknown whether the myelination of PV + interneurons is also modulated by intrinsic activity. Here, we utilized cell-type specific Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in adult male and female mice to activate a sparse population of medial prefrontal cortex PV + interneurons. Using single-cell axonal reconstructions, we find that DREADD-stimulated PV + interneurons exhibit a nearly two-fold increase in total length of myelination, predominantly mediated by a parallel increase of axonal arborization and number of internodes. In contrast, the distribution of axonal inter-branch segment distance and myelin internode length were not significantly altered. Topographical analysis revealed that myelination of DREADD-stimulated cells extended to higher axonal branch orders, while retaining a similar inter-branch distance threshold for myelination. Together, our results demonstrate that chemogenetically-induced neuronal activity increases the myelination of neocortical PV + interneurons mediated at least in part by an elaboration of their axonal morphology. SIGNIFICANCE STATEMENT Myelination is the wrapping of an axon in order to optimize conduction velocity in an energy-efficient manner. Previous studies have shown that myelination of neocortical pyramidal neurons is experience and activity-dependent. We now show that activity-dependent myelin plasticity in the adult neocortex extends to parvalbumin-expressing fast-spiking interneurons. Specifically, chemogenetic stimulation of parvalbumin interneurons in the medial prefrontal cortex significantly enhanced axonal myelination, which was paralleled by an increase in axonal arborization. This suggests that activity

  5. Axon Counts Yield Multiple Options for Triceps Fascicular Nerve to Axillary Nerve Transfer. (United States)

    Khair, M Michael; Schreiber, Joseph J; Rosenblatt, Lauren; Byun, David J; Lee, Steve K; Wolfe, Scott W


    To evaluate the relative axonal match between potential donor and recipient nerves, so that maximal reinnervation potential may be reached with the least chance of donor site morbidity. In 10 fresh-frozen cadaveric specimens, the main trunk and anterior, posterior, sensory and teres minor branches of the axillary nerve were identified, as were the radial nerve branches to the long, medial, and lateral heads of the triceps. The swing distances of the triceps fascicular nerve branches and the axillary nerve branches relative to the inferior border of the teres major muscle were recorded. Histomorphological analysis and axon counts were performed on sections of each branch. The median number of axons in the main axillary trunk was 7,887, with 4,052, 1,242, and 1,161 axons in the anterior, posterior, and teres minor branches, respectively. All specimens had a single long head triceps branch (median, 2,302 axons), a range of 1 to 3 branches to the medial head of the triceps (composite axon count, 2,198 axons), and 1 to 3 branches to the lateral head of the triceps (composite average, 1,462 axons). The medial and lateral head branches had sufficient swing distance to reach the anterior branch of the axillary nerve in all 10 specimens, with only 4 specimens having adequate long head branch swing distances. It is anatomically feasible to transfer multiple branches of the radial nerve supplying the medial, lateral, and sometimes, long head of the triceps to all branches of the axillary nerve in an attempt to reinnervate the deltoid and teres minor muscles. Understanding the axon counts of the different possible transfer combinations will improve operative flexibility and enable peripheral nerve surgeons to reinnervate for both abduction and external rotation with the highest donor/recipient axon count ratios. Copyright © 2016 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

  6. Dorsal column sensory axons degenerate due to impaired microvascular perfusion after spinal cord injury in rats (United States)

    Muradov, Johongir M.; Ewan, Eric E.; Hagg, Theo


    The mechanisms contributing to axon loss after spinal cord injury (SCI) are largely unknown but may involve microvascular loss as we have previously suggested. Here, we used a mild contusive injury (120 kdyn IH impactor) at T9 in rats focusing on ascending primary sensory dorsal column axons, anterogradely traced from the sciatic nerves. The injury caused a rapid and progressive loss of dorsal column microvasculature and oligodendrocytes at the injury site and penumbra and a ~70% loss of the sensory axons, by 24 hours. To model the microvascular loss, focal ischemia of the T9 dorsal columns was achieved via phototoxic activation of intravenously injected rose bengal. This caused an ~53% loss of sensory axons and an ~80% loss of dorsal column oligodendrocytes by 24 hours. Axon loss correlated with the extent and axial length of microvessel and oligodendrocyte loss along the dorsal column. To determine if oligodendrocyte loss contributes to axon loss, the glial toxin ethidium bromide (EB; 0.3 µg/µl) was microinjected into the T9 dorsal columns, and resulted in an ~88% loss of dorsal column oligodendrocytes and an ~56% loss of sensory axons after 72 hours. EB also caused an ~72% loss of microvessels. Lower concentrations of EB resulted in less axon, oligodendrocyte and microvessel loss, which were highly correlated (R2 = 0.81). These data suggest that focal spinal cord ischemia causes both oligodendrocyte and axon degeneration, which are perhaps linked. Importantly, they highlight the need of limiting the penumbral spread of ischemia and oligodendrocyte loss after SCI in order to protect axons. PMID:23978615

  7. Dihydrotestosterone ameliorates degeneration in muscle, axons and motoneurons and improves motor function in amyotrophic lateral sclerosis model mice.

    Directory of Open Access Journals (Sweden)

    Young-Eun Yoo

    Full Text Available Amyotrophic lateral sclerosis (ALS is a lethal disease characterized by a progressive loss of motoneurons. The clinical symptoms include skeletal muscle weakness and atrophy, which impairs motor performance and eventually leads to respiratory failure. We tested whether dihydrotestosterone (DHT, which has both anabolic effects on muscle and neuroprotective effects on axons and motoneurons, can ameliorate clinical symptoms in ALS. A silastic tube containing DHT crystals was implanted subcutaneously in SOD1-G93A mice at early symptomatic age when decreases in body weight and grip-strength were observed as compared to wild-type mice. DHT-treated SOD1-G93A mice demonstrated ameliorated muscle atrophy and increased body weight, which was associated with stronger grip-strength. DHT treatment increased the expression of insulin-like growth factor-1 in muscle, which can exert myotrophic as well as neurotrophic effects through retrograde transport. DHT treatment attenuated neuromuscular junction denervation, and axonal and motoneuron loss. DHT-treated SOD1-G93A mice demonstrated improvement in motor behavior as assessed by rota-rod and gait analyses, and an increased lifespan. Application of DHT is a relatively simple and non-invasive procedure, which may be translated into therapy to improve the quality of life for ALS patients.

  8. Bicyclic-Capped Histone Deacetylase 6 Inhibitors with Improved Activity in a Model of Axonal Charcot-Marie-Tooth Disease. (United States)

    Shen, Sida; Benoy, Veronick; Bergman, Joel A; Kalin, Jay H; Frojuello, Mariana; Vistoli, Giulio; Haeck, Wanda; Van Den Bosch, Ludo; Kozikowski, Alan P


    Charcot-Marie-Tooth (CMT) disease is a disorder of the peripheral nervous system where progressive degeneration of motor and sensory nerves leads to motor problems and sensory loss and for which no pharmacological treatment is available. Recently, it has been shown in a model for the axonal form of CMT that histone deacetylase 6 (HDAC6) can serve as a target for the development of a pharmacological therapy. Therefore, we aimed at developing new selective and activity-specific HDAC6 inhibitors with improved biochemical properties. By utilizing a bicyclic cap as the structural scaffold from which to build upon, we developed several analogues that showed improved potency compared to tubastatin A while maintaining excellent selectivity compared to HDAC1. Further screening in N2a cells examining both the acetylation of α-tubulin and histones narrowed down the library of compounds to three potent and selective HDAC6 inhibitors. In mutant HSPB1-expressing DRG neurons, serving as an in vitro model for CMT2, these inhibitors were able to restore the mitochondrial axonal transport deficits. Combining structure-based development of HDAC6 inhibitors, screening in N2a cells and in a neuronal model for CMT2F, and preliminary ADMET and pharmacokinetic profiles, resulted in the selection of compound 23d that possesses improved biochemical, functional, and druglike properties compared to tubastatin A.

  9. A phosphorylation cascade controls the degradation of active SREBP1. (United States)

    Bengoechea-Alonso, Maria T; Ericsson, Johan


    Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

  10. Tyrosine phosphorylation of Rab7 by Src kinase. (United States)

    Lin, Xiaosi; Zhang, Jiaming; Chen, Lingqiu; Chen, Yongjun; Xu, Xiaohui; Hong, Wanjin; Wang, Tuanlao


    The small molecular weight GTPase Rab7 is a key regulator for late endosomal/lysosomal membrane trafficking, it was known that Rab7 is phosphorylated, but the corresponding kinase and the functional regulation of Rab7 phosphorylation remain unclear. We provide evidence here that Rab7 is a substrate of Src kinase, and is tyrosine-phosphorylated by Src, withY183 residue of Rab7 being the optimal phosphorylation site for Src. Further investigations demonstrated that the tyrosine phosphorylation of Rab7 depends on the guanine nucleotide binding activity of Rab7 and the activity of Src kinase. The tyrosine phosphorylation of Rab7 is physiologically induced by EGF, and impairs the interaction of Rab7 with RILP, consequently inhibiting EGFR degradation and sustaining Akt signaling. These results suggest that the tyrosine phosphorylation of Rab7 may be involved in coordinating membrane trafficking and cell signaling. Copyright © 2017. Published by Elsevier Inc.

  11. The axonal guidance receptor neogenin promotes acute inflammation.

    Directory of Open Access Journals (Sweden)

    Klemens König

    Full Text Available Neuronal guidance proteins (NGP were originally described in the context of axonal growth and migration. Yet recent work has demonstrated that NGPs also serve as guidance cues for immune competent cells. A crucial target receptor for NGPs during embryonic development is the neogenin receptor, however its role during acute inflammation is unknown. We report here that neogenin is abundantly expressed outside the nervous system and that animals with endogenous repression of neogenin (Neo1(-/- demonstrate attenuated changes of acute inflammation. Studies using functional inhibition of neogenin resulted in a significant attenuation of inflammatory peritonitis. In studies employing bone marrow chimeric animals we found the hematopoietic presence of Neo1(-/- to be responsible for the attenuated inflammatory response. Taken together our studies suggest that the guidance receptor neogenin holds crucial importance for the propagation of an acute inflammatory response and further define mechanisms shared between the nervous and the immune system.

  12. Video Object Tracking in Neural Axons with Fluorescence Microscopy Images

    Directory of Open Access Journals (Sweden)

    Liang Yuan


    tracking. In this paper, we describe two automated tracking methods for analyzing neurofilament movement based on two different techniques: constrained particle filtering and tracking-by-detection. First, we introduce the constrained particle filtering approach. In this approach, the orientation and position of a particle are constrained by the axon’s shape such that fewer particles are necessary for tracking neurofilament movement than object tracking techniques based on generic particle filtering. Secondly, a tracking-by-detection approach to neurofilament tracking is presented. For this approach, the axon is decomposed into blocks, and the blocks encompassing the moving neurofilaments are detected by graph labeling using Markov random field. Finally, we compare two tracking methods by performing tracking experiments on real time-lapse image sequences of neurofilament movement, and the experimental results show that both methods demonstrate good performance in comparison with the existing approaches, and the tracking accuracy of the tracing-by-detection approach is slightly better between the two.

  13. Phosphoryl functionalized mesoporous silica for uranium adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Guo; Yurun, Feng; Li, Ma; Dezhi, Gao; Jie, Jing; Jincheng, Yu; Haibin, Sun [Key Laboratory for Liquid-Solid Structural Evolution & Processing of Materials of Ministry of Education, Shandong University, Jinan 250061 (China); Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, Shandong University, Jinan 250061 (China); Hongyu, Gong, E-mail: [Key Laboratory for Liquid-Solid Structural Evolution & Processing of Materials of Ministry of Education, Shandong University, Jinan 250061 (China); Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, Shandong University, Jinan 250061 (China); Yujun, Zhang, E-mail: [Key Laboratory for Liquid-Solid Structural Evolution & Processing of Materials of Ministry of Education, Shandong University, Jinan 250061 (China); Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, Shandong University, Jinan 250061 (China)


    Highlights: • Phosphoryl functionalized mesoporous silica (TBP-SBA-15) is synthesized. • The amino and phosphoryl groups are successfully grafted on SBA-15. • TBP-SBA-15 has high and rapid uranium adsorption capacity in broad pH range. • The U(VI) adsorption of TBP-SBA-15 is spontaneous and belongs to chemical adsorption. - Abstract: Phosphoryl functionalized mesoporous silica (TBP-SBA-15) was synthesized by modified mesoporous silica with γ-amino propyl triethoxy silane and tributyl phosphate. The obtained samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray diffraction (SAXRD), thermo-gravimetric/differential thermalanalyzer (TG/DTA), N{sub 2} adsorption–desorption (BET) and Fourier transform infrared spectroscopy (FT-IR) techniques. Results showed that TBP-SBA-15 had large surface areas with ordered channel structure. Moreover, the effects of adsorption time, sorbent dose, solution pH, initial uranium concentration and temperature on the uranium adsorption behaviors were investigated. TBP-SBA-15 showed a high uranium adsorption capacity in a broad range of pH values. The U(VI) adsorption rate of TBP-SBA-15 was fast and nearly achieved completion in 10 min with the sorbent dose of 1 g/L. The U(VI) adsorption of TBP-SBA-15 followed the pseudo-second-order kinetic model and Freundlich isotherm model, indicating that the process was belonged to chemical adsorption. Furthermore, the thermodynamic parameters (ΔG{sup 0}, ΔH{sup 0} and ΔS{sup 0}) confirmed that the adsorption process was endothermic and spontaneous.

  14. Ephexin1 Is Required for Eph-Mediated Limb Trajectory of Spinal Motor Axons. (United States)

    Chang, Chih-Ju; Chang, Ming-Yuan; Chou, Szu-Yi; Huang, Chi-Chen; Chuang, Jian-Ying; Hsu, Tsung-I; Chang, Hsing-Fang; Wu, Yi-Hsin; Wu, Chung-Che; Morales, Daniel; Kania, Artur; Kao, Tzu-Jen


    The precise assembly of a functional nervous system relies on the guided migration of axonal growth cones, which is made possible by signals transmitted to the cytoskeleton by cell surface-expressed guidance receptors. We investigated the function of ephexin1, a Rho guanine nucleotide exchange factor, as an essential growth-cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show that ephexin1 is expressed in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Ephexin1 loss of function and gain of function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of ephexin1 in motor axon guidance. In addition, ephexin1 loss in mice of either sex led to LMC axon trajectory selection errors. We also show that ephexin1 knockdown attenuates the growth preference of LMC neurites against ephrins in vitro and Eph receptor-mediated retargeting of LMC axons in vivo , suggesting that ephexin1 is required in Eph-mediated LMC motor axon guidance. Finally, both ephexin1 knockdown and ectopic expression of nonphosphorylatable ephexin1 mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating ephexin1 as an Src target in Eph signal relay in this context. In summary, our findings demonstrate that ephexin1 is essential for motor axon guidance and suggest an important role in relaying ephrin:Eph signals that mediate motor axon trajectory selection. SIGNIFICANCE STATEMENT The proper development of functioning neural circuits requires precise nerve connections among neurons or between neurons and their muscle targets. The Eph tyrosine kinase receptors expressed in neurons are important in many contexts during neural-circuit formation, such as axon outgrowth, axon guidance, and synaptic formation, and have been

  15. Neuron Morphology Influences Axon Initial Segment Plasticity123 (United States)


    In most vertebrate neurons, action potentials are initiated in the axon initial segment (AIS), a specialized region of the axon containing a high density of voltage-gated sodium and potassium channels. It has recently been proposed that neurons use plasticity of AIS length and/or location to regulate their intrinsic excitability. Here we quantify the impact of neuron morphology on AIS plasticity using computational models of simplified and realistic somatodendritic morphologies. In small neurons (e.g., dentate granule neurons), excitability was highest when the AIS was of intermediate length and located adjacent to the soma. Conversely, neurons having larger dendritic trees (e.g., pyramidal neurons) were most excitable when the AIS was longer and/or located away from the soma. For any given somatodendritic morphology, increasing dendritic membrane capacitance and/or conductance favored a longer and more distally located AIS. Overall, changes to AIS length, with corresponding changes in total sodium conductance, were far more effective in regulating neuron excitability than were changes in AIS location, while dendritic capacitance had a larger impact on AIS performance than did dendritic conductance. The somatodendritic influence on AIS performance reflects modest soma-to-AIS voltage attenuation combined with neuron size-dependent changes in AIS input resistance, effective membrane time constant, and isolation from somatodendritic capacitance. We conclude that the impact of AIS plasticity on neuron excitability will depend largely on somatodendritic morphology, and that, in some neurons, a shorter or more distally located AIS may promote, rather than limit, action potential generation. PMID:27022619

  16. Multichannel activity propagation across an engineered axon network (United States)

    Chen, H. Isaac; Wolf, John A.; Smith, Douglas H.


    Objective. Although substantial progress has been made in mapping the connections of the brain, less is known about how this organization translates into brain function. In particular, the massive interconnectivity of the brain has made it difficult to specifically examine data transmission between two nodes of the connectome, a central component of the ‘neural code.’ Here, we investigated the propagation of multiple streams of asynchronous neuronal activity across an isolated in vitro ‘connectome unit.’ Approach. We used the novel technique of axon stretch growth to create a model of a long-range cortico-cortical network, a modular system consisting of paired nodes of cortical neurons connected by axon tracts. Using optical stimulation and multi-electrode array recording techniques, we explored how input patterns are represented by cortical networks, how these representations shift as they are transmitted between cortical nodes and perturbed by external conditions, and how well the downstream node distinguishes different patterns. Main results. Stimulus representations included direct, synaptic, and multiplexed responses that grew in complexity as the distance between the stimulation source and recorded neuron increased. These representations collapsed into patterns with lower information content at higher stimulation frequencies. With internodal activity propagation, a hierarchy of network pathways, including latent circuits, was revealed using glutamatergic blockade. As stimulus channels were added, divergent, non-linear effects were observed in local versus distant network layers. Pairwise difference analysis of neuronal responses suggested that neuronal ensembles generally outperformed individual cells in discriminating input patterns. Significance. Our data illuminate the complexity of spiking activity propagation in cortical networks in vitro, which is characterized by the transformation of an input into myriad outputs over several network layers

  17. Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III (United States)

    Udina, Esther; Putman, Charles T.; Harris, Luke R.; Tyreman, Neil; Cook, Victoria E.


    Key points Smn +/− transgenic mouse is a model of the mildest form of spinal muscular atrophy.Although there is a loss of spinal motoneurons in 11‐month‐old animals, muscular force is maintained.This maintained muscular force is mediated by reinnervation of the denervated fibres by surviving motoneurons.The spinal motoneurons in these animals do not show an increased susceptibility to death after nerve injury and they retain their regenerative capacity.We conclude that the hypothesized immaturity of the neuromuscular system in this model cannot explain the loss of motoneurons by systematic die‐back. Abstract Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and is the leading genetic cause of infantile death. Patients lack the SMN1 gene with the severity of the disease depending on the number of copies of the highly homologous SMN2 gene. Although motoneuron death in the Smn +/− transgenic mouse model of the mildest form of SMA, SMA type III, has been reported, we have used retrograde tracing of sciatic and femoral motoneurons in the hindlimb with recording of muscle and motor unit isometric forces to count the number of motoneurons with intact neuromuscular connections. Thereby, we investigated whether incomplete maturation of the neuromuscular system induced by survival motoneuron protein (SMN) defects is responsible for die‐back of axons relative to survival of motoneurons. First, a reduction of ∼30% of backlabelled motoneurons began relatively late, at 11 months of age, with a significant loss of 19% at 7 months. Motor axon die‐back was affirmed by motor unit number estimation. Loss of functional motor units was fully compensated by axonal sprouting to retain normal contractile force in four hindlimb muscles (three fast‐twitch and one slow‐twitch) innervated by branches of the sciatic nerve. Second, our evaluation of whether axotomy of motoneurons in the adult Smn +/− transgenic mouse increases their

  18. Compensatory axon sprouting for very slow axonal die-back in a transgenic model of spinal muscular atrophy type III. (United States)

    Udina, Esther; Putman, Charles T; Harris, Luke R; Tyreman, Neil; Cook, Victoria E; Gordon, Tessa


    Smn +/- transgenic mouse is a model of the mildest form of spinal muscular atrophy. Although there is a loss of spinal motoneurons in 11-month-old animals, muscular force is maintained. This maintained muscular force is mediated by reinnervation of the denervated fibres by surviving motoneurons. The spinal motoneurons in these animals do not show an increased susceptibility to death after nerve injury and they retain their regenerative capacity. We conclude that the hypothesized immaturity of the neuromuscular system in this model cannot explain the loss of motoneurons by systematic die-back. Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and is the leading genetic cause of infantile death. Patients lack the SMN1 gene with the severity of the disease depending on the number of copies of the highly homologous SMN2 gene. Although motoneuron death in the Smn +/- transgenic mouse model of the mildest form of SMA, SMA type III, has been reported, we have used retrograde tracing of sciatic and femoral motoneurons in the hindlimb with recording of muscle and motor unit isometric forces to count the number of motoneurons with intact neuromuscular connections. Thereby, we investigated whether incomplete maturation of the neuromuscular system induced by survival motoneuron protein (SMN) defects is responsible for die-back of axons relative to survival of motoneurons. First, a reduction of ∼30% of backlabelled motoneurons began relatively late, at 11 months of age, with a significant loss of 19% at 7 months. Motor axon die-back was affirmed by motor unit number estimation. Loss of functional motor units was fully compensated by axonal sprouting to retain normal contractile force in four hindlimb muscles (three fast-twitch and one slow-twitch) innervated by branches of the sciatic nerve. Second, our evaluation of whether axotomy of motoneurons in the adult Smn +/- transgenic mouse increases their susceptibility to cell death demonstrated

  19. Regulation of auxin transport during gravitropism (United States)

    Rashotte, A.; Brady, S.; Kirpalani, N.; Buer, C.; Muday, G.

    Plants respond to changes in the gravity vector by differential growth across the gravity-stimulated organ. The plant hormone auxin, which is normally basipetally transported, changes in direction and auxin redistribution has been suggested to drive this differential growth or gravitropism. The mechanisms by which auxin transport directionality changes in response to a change in gravity vector are largely unknown. Using the model plant, Arabidopsis thaliana, we have been exploring several regulatory mechanisms that may control auxin transport. Mutations that alter protein phosphorylation suggest that auxin transport in arabidopsis roots may be controlled via phosphorylation and this signal may facilitate gravitropic bending. The protein kinase mutant pinoid (pid9) has reduced auxin transport; whereas the protein phosphatase mutant, rcn1, has elevated transport, suggesting reciprocal regulation of auxin transport by reversible protein phosphorylation. In both of these mutants, the auxin transport defects are accompanied by gravitropic defects, linking phosphorylation signaling to gravity-induced changes in auxin transport. Additionally, auxin transport may be regulated during gravity response by changes in an endogenous auxin efflux inhibitor. Flavonoids, such as quercetin and kaempferol, have been implicated in regulation of auxin transport in vivo and in vitro. Mutants that make no flavonoids have reduced root gravitropic bending. Furthermore, changes in auxin-induced gene expression and flavonoid accumulation patterns have been observed during gravity stimulation. Current studies are examining whether there are spatial and temporal changes in flavonoid accumulation that precede gravitropic bending and whether the absence of these changes are the cause of the altered gravity response in plants with mutations that block flavonoid synthesis. These results support the idea that auxin transport may be regulated during gravity response by several mechanisms including

  20. Mitochondrial dysfunction and oxidative damage cooperatively fuel axonal degeneration in X-linked adrenoleukodystrophy. (United States)

    Fourcade, Stéphane; López-Erauskin, Jone; Ruiz, Montserrat; Ferrer, Isidre; Pujol, Aurora


    X-linked adrenoleukodystrophy (X-ALD) is the most frequent inherited monogenic demyelinating disease (minimal incidence 1:17,000). It is often lethal and currently lacks a satisfactory therapy. The disease is caused by loss of function of the ABCD1 gene, a peroxisomal ATP-binding cassette transporter, resulting in the accumulation of VLCFA (very long-chain fatty acids) in organs and plasma. Understanding of the aetiopathogenesis is a prerequisite for the development of novel therapeutic strategies. Functional genomics analysis of an ABCD1 null mouse, a mouse model for adrenomyeloneuropathy, has revealed presymptomatic alterations in several metabolic pathways converging on redox and bioenergetic homeostasis, with failure of mitochondrial OXPHOS disruption and mitochondrial depletion. These defects could be major contributors to the neurodegenerative cascade, as has been reported in several neurodegenerative disorders. Drugs targeting the redox imbalance/mitochondria dysfunction interplay have shown efficacy at halting axonal degeneration and associated disability in the mouse, and thus offer therapeutic hope. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  1. Mitosis in neurons: Roughex and APC/C maintain cell cycle exit to prevent cytokinetic and axonal defects in Drosophila photoreceptor neurons.

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    Robert Ruggiero

    Full Text Available The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.

  2. SH3 domain tyrosine phosphorylation--sites, role and evolution.

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    Zuzana Tatárová

    Full Text Available BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F. This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

  3. Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy. (United States)

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey


    Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

  4. Glia initiate brain assembly through non-canonical Chimaerin/Furin axon guidance in C. elegans (United States)

    Rapti, Georgia; Li, Chang; Shan, Alan; Lu, Yun; Shaham, Shai


    Brain assembly is hypothesized to begin when pioneer axons extend over non-neuronal cells, forming tracts guiding follower axons. Yet pioneer-neuron identities, their guidance substrates, and their interactions, are not well understood. Here, using time-lapse embryonic imaging, genetics, protein-interaction, and functional studies, we uncover the early events of C. elegans brain assembly. We demonstrate that C. elegans glia are key for assembly initiation, guiding pioneer and follower axons using distinct signals. Pioneer sublateral neurons, with unique growth properties, anatomy, and innervation, cooperate with glia to mediate follower-axon guidance. We further identify a CHIN-1/Chimaerin-KPC-1/Furin double mutant that severely disrupts assembly. CHIN-1/Chimaerin and KPC-1/Furin function non-canonically in glia and pioneer neurons for guidance-cue trafficking. We exploit this bottleneck to define roles for glial Netrin and Semaphorin in pioneer- and follower-axon guidance, respectively, and for glial and pioneer-neuron Flamingo/CELSR in follower-axon navigation. Altogether, our studies reveal previously-unknown glial roles in pioneer-axon guidance, suggesting conserved brain-assembly principles. PMID:28846083

  5. Expression of plasminogen activator inhibitor-1 by olfactory ensheathing glia promotes axonal regeneration. (United States)

    Simón, Diana; Martín-Bermejo, Maria Jesús; Gallego-Hernández, Maria Teresa; Pastrana, Erika; García-Escudero, Vega; García-Gómez, Ana; Lim, Filip; Díaz-Nido, Javier; Avila, Jesús; Moreno-Flores, Maria Teresa


    Olfactory ensheathing glia (OEG) cells are known to facilitate repair following axotomy of adult neurons, although the molecular mechanisms involved are not fully understood. We previously identified plasminogen activator inhibitor-1 (PAI-1), proteinase-activated receptor-1 (PAR-1), and thrombomodulin (TM) as candidates to regulate rat OEG-dependent axonal regeneration. In this study, we have validated the involvement of these proteins in promoting axonal regeneration by immortalized human OEGs. We studied the effect of silencing these proteins in OEGs on their capacity to promote the regeneration of severed adult retinal ganglion cells (RGCs) axons. Our results support the role of glial PAI-1 as a downstream effector of PAR-1 in promoting axon regeneration. In contrast, we found that TM inhibits OEG induced-axonal regeneration. We also assessed the signaling pathways downstream of PAR-1 that might modulate PAI-1 expression, observing that specifically inhibiting Gα(i), Rho kinase, or PLC and PKC downregulated the expression of PAI-1 in OEGs, with a concomitant reduction in OEG-dependent axon regeneration in adult RGCs. Our findings support an important role for the thrombin system in regulating adult axonal regeneration by OEGs. Copyright © 2011 Wiley-Liss, Inc.

  6. The Influence of Glutamate on Axonal Compound Action Potential In Vitro. (United States)

    Abouelela, Ahmed; Wieraszko, Andrzej


    Background  Our previous experiments demonstrated modulation of the amplitude of the axonal compound action potential (CAP) by electrical stimulation. To verify assumption that glutamate released from axons could be involved in this phenomenon, the modification of the axonal CAP induced by glutamate was investigated. Objectives  The major objective of this research is to verify the hypothesis that axonal activity would trigger the release of glutamate, which in turn would interact with specific axonal receptors modifying the amplitude of the action potential. Methods  Segments of the sciatic nerve were exposed to exogenous glutamate in vitro, and CAP was recorded before and after glutamate application. In some experiments, the release of radioactive glutamate analog from the sciatic nerve exposed to exogenous glutamate was also evaluated. Results  The glutamate-induced increase in CAP was blocked by different glutamate receptor antagonists. The effect of glutamate was not observed in Ca-free medium, and was blocked by antagonists of calcium channels. Exogenous glutamate, applied to the segments of sciatic nerve, induced the release of radioactive glutamate analog, demonstrating glutamate-induced glutamate release. Immunohistochemical examination revealed that axolemma contains components necessary for glutamatergic neurotransmission. Conclusion  The proteins of the axonal membrane can under the influence of electrical stimulation or exogenous glutamate change membrane permeability and ionic conductance, leading to a change in the amplitude of CAP. We suggest that increased axonal activity leads to the release of glutamate that results in changes in the amplitude of CAPs.

  7. Optogenetically enhanced axon regeneration: motor versus sensory neuron-specific stimulation. (United States)

    Ward, Patricia J; Clanton, Scott L; English, Arthur W


    Brief neuronal activation in injured peripheral nerves is both necessary and sufficient to enhance motor axon regeneration, and this effect is specific to the activated motoneurons. It is less clear whether sensory neurons respond in a similar manner to neuronal activation following peripheral axotomy. Further, it is unknown to what extent enhancement of axon regeneration with increased neuronal activity relies on a reflexive interaction within the spinal circuitry. We used mouse genetics and optical tools to evaluate the precision and selectivity of system-specific neuronal activation to enhance axon regeneration in a mixed nerve. We evaluated sensory and motor axon regeneration in two different mouse models expressing the light-sensitive cation channel, channelrhodopsin (ChR2). We selectively activated either sensory or motor axons using light stimulation combined with transection and repair of the sciatic nerve. Regardless of genotype, the number of ChR2-positive neurons whose axons had regenerated successfully was greater following system-specific optical treatment, with no effect on the number of ChR2-negative neurons (whether motor or sensory neurons). We conclude that acute system-specific neuronal activation is sufficient to enhance both motor and sensory axon regeneration. This regeneration-enhancing effect is likely cell autonomous. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  8. Modeling the mechanics of axonal fiber tracts using the embedded finite element method. (United States)

    Garimella, Harsha T; Kraft, Reuben H


    A subject-specific human head finite element model with embedded axonal fiber tractography obtained from diffusion tensor imaging was developed. The axonal fiber tractography finite element model was coupled with the volumetric elements in the head model using the embedded element method. This technique enables the calculation of axonal strains and real-time tracking of the mechanical response of the axonal fiber tracts. The coupled model was then verified using pressure and relative displacement-based (between skull and brain) experimental studies and was employed to analyze a head impact, demonstrating the applicability of this method in studying axonal injury. Following this, a comparison study of different injury criteria was performed. This model was used to determine the influence of impact direction on the extent of the axonal injury. The results suggested that the lateral impact loading is more dangerous compared to loading in the sagittal plane, a finding in agreement with previous studies. Through this analysis, we demonstrated the viability of the embedded element method as an alternative numerical approach for studying axonal injury in patient-specific human head models. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Characterizing Semaphorin-Mediated Effects on Sensory and Motor Axon Pathfinding and Connectivity During Embryonic Development. (United States)

    Huettl, Rosa Eva; Huber, Andrea B


    How are precise connectivity to peripheral targets and corresponding sensory-motor networks established during developmental innervation of the vertebrate extremities? The formation of functional sensory-motor circuits requires highly appropriate temporal and spatial regulation of axon growth which is achieved through the combination of different molecular mechanisms such as communication between heterotypic fiber systems, axon-environment, or axon-glia interactions that ensure proper fasciculation and accurate pathfinding to distal targets. Family members of the class 3 semaphorins and their cognate receptors, the neuropilins, were shown to govern various events during wiring of central and peripheral circuits, with mice lacking Sema3-Npn signaling showing deficits in timing of growth, selective fasciculation, guidance fidelity, and coupling of sensory axon growth to motor axons at developmental time points. Given the accuracy with which these processes have to interact in a stepwise manner, deficiency of the smallest cog in the wheel may impact severely on the faithful establishment and functionality of peripheral circuitries, ultimately leading to behavioral impairments or even cause the death of the animal. Reliable quantitative analyses of sensory-motor fasciculation, extension, and guidance of axons to their cognate target muscles and the skin during development, but also assessment of physiological and behavioral consequences at adult age, are therefore a necessity to extend our understanding of the molecular mechanisms of peripheral circuit formation. In this chapter we provide a detailed methodology to characterize class 3 semaphorin-mediated effects on peripheral sensory and motor axon pathfinding and connectivity during embryonic development.

  10. Non-nuclear Pool of Splicing Factor SFPQ Regulates Axonal Transcripts Required for Normal Motor Development. (United States)

    Thomas-Jinu, Swapna; Gordon, Patricia M; Fielding, Triona; Taylor, Richard; Smith, Bradley N; Snowden, Victoria; Blanc, Eric; Vance, Caroline; Topp, Simon; Wong, Chun-Hao; Bielen, Holger; Williams, Kelly L; McCann, Emily P; Nicholson, Garth A; Pan-Vazquez, Alejandro; Fox, Archa H; Bond, Charles S; Talbot, William S; Blair, Ian P; Shaw, Christopher E; Houart, Corinne


    Recent progress revealed the complexity of RNA processing and its association to human disorders. Here, we unveil a new facet of this complexity. Complete loss of function of the ubiquitous splicing factor SFPQ affects zebrafish motoneuron differentiation cell autonomously. In addition to its nuclear localization, the protein unexpectedly localizes to motor axons. The cytosolic version of SFPQ abolishes motor axonal defects, rescuing key transcripts, and restores motility in the paralyzed sfpq null mutants, indicating a non-nuclear processing role in motor axons. Novel variants affecting the conserved coiled-coil domain, so far exclusively found in fALS exomes, specifically affect the ability of SFPQ to localize in axons. They broadly rescue morphology and motility in the zebrafish mutant, but alter motor axon morphology, demonstrating functional requirement for axonal SFPQ. Altogether, we uncover the axonal function of the splicing factor SFPQ in motor development and highlight the importance of the coiled-coil domain in this process. VIDEO ABSTRACT. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization. (United States)

    Hummel, T; Leifker, K; Klämbt, C


    In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. Here, we show that the gene kette is required for the normal projection of the VUM axons and subsequently disrupts glial migration. Axonal projection defects are also found for many other moto- and interneurons. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The KETTE protein is homologous to the transmembrane protein HEM-2/NAP1 evolutionary conserved from worms to vertebrates. In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2-SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, we show that kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins RAC1 and CDC42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant DCDC42 or DRAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated DRAC1 transgene. Our data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding.

  12. GDF10 Is a Signal for Axonal Sprouting and Functional Recovery after Stroke (United States)

    Li, S; Nie, EH; Yin, Y; Benowitz, LI; Tung, S; Vinters, HV; Bahjat, FR; Stenzel-Poore, MP; Kawaguchi, R; Coppola, G; Carmichael, ST


    Stroke produces a limited process of neural repair. Axonal sprouting in cortex adjacent to the infarct is part of this recovery process, but the signal that initiates axonal sprouting is not known. Growth and Differentiation Factor 10 (GDF10) is induced in peri-infarct neurons in mouse, non-human primate and human. GDF10 promotes axonal outgrowth in vitro in mouse, rat and human neurons through TGFβRI/II signaling. Using pharmacogenetic gain and loss of function studies, GDF10 produces axonal sprouting and enhanced functional recovery after stroke; knocking down GDF10 blocks axonal sprouting and reduces recovery. RNA-seq from peri-infarct cortical neurons indicates that GDF10 downregulates PTEN and upregulates PI3 kinase signaling and induces specific axonal guidance molecules. Unsupervised genome-wide association analysis of the GDF10 transcriptome shows that it is not related to neurodevelopment but may partially overlap with other CNS injury patterns. GDF10 is a stroke-induced signal for axonal sprouting and functional recovery. PMID:26502261

  13. Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade

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    Alexandre Dumoulin


    Full Text Available Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP, the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα. In the absence of any one of these components, neurons in dorsal root ganglia (DRG and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

  14. Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44

    International Nuclear Information System (INIS)

    Alvisi, Gualtiero; Marin, Oriano; Pari, Gregory; Mancini, Manuela; Avanzi, Simone; Loregian, Arianna; Jans, David A.; Ripalti, Alessandro


    The processivity factor of human cytomegalovirus DNA polymerase, phosphoprotein ppUL44, is essential for viral replication. During viral infection ppUL44 is phosphorylated by the viral kinase pUL97, but neither the target residues on ppUL44 nor the effect of phosphorylation on ppUL44's activity are known. We report here that ppUL44 is phosphorylated when transiently expressed in mammalian cells and coimmunoprecipitates with cellular kinases. Of three potential phosphorylation sites (S413, S415, S418) located upstream of ppUL44's nuclear localization signal (NLS) and one (T427) within the NLS itself, protein kinase CK2 (CK2) specifically phosphorylates S413, to trigger a cascade of phosphorylation of S418 and S415 by CK1 and CK2, respectively. Negative charge at the CK2/CK1 target serine residues facilitates optimal nuclear accumulation of ppUL44, whereas negative charge on T427, a potential cyclin-dependent 1 phosphorylation site, strongly decreases nuclear accumulation. Thus, nuclear transport of ppUL44 is finely tuned during viral infection through complex phosphorylation events.

  15. Extracellular matrix molecules play diverse roles in the growth and guidance of central nervous system axons

    Directory of Open Access Journals (Sweden)

    M.A. Pires-Neto


    Full Text Available Axon growth and guidance represent complex biological processes in which probably intervene diverse sets of molecular cues that allow for the appropriate wiring of the central nervous system (CNS. The extracellular matrix (ECM represents a major contributor of molecular signals either diffusible or membrane-bound that may regulate different stages of neural development. Some of the brain ECM molecules form tridimensional structures (tunnels and boundaries that appear during time- and space-regulated events, possibly playing relevant roles in the control of axon elongation and pathfinding. This short review focuses mainly on the recognized roles played by proteoglycans, laminin, fibronectin and tenascin in axonal development during ontogenesis.

  16. Axonal and presynaptic protein synthesis: new insights into the biology of the neuron. (United States)

    Giuditta, Antonio; Kaplan, Barry B; van Minnen, Jan; Alvarez, Jaime; Koenig, Edward


    The presence of a local mRNA translation system in axons and terminals was proposed almost 40 years ago. Over the ensuing period, an impressive body of evidence has grown to support this proposal -- yet the nerve cell body is still considered to be the only source of axonal and presynaptic proteins. To dispel this lingering neglect, we now present the wealth of recent observations bearing on this central idea, and consider their impact on our understanding of the biology of the neuron. We demonstrate that extrasomatic translation sites, which are now well recognized in dendrites, are also present in axonal and presynaptic compartments.

  17. Current Opportunities for Clinical Monitoring of Axonal Pathology in Traumatic Brain Injury

    Directory of Open Access Journals (Sweden)

    Parmenion P. Tsitsopoulos


    Full Text Available Traumatic brain injury (TBI is a multidimensional and highly complex disease commonly resulting in widespread injury to axons, due to rapid inertial acceleration/deceleration forces transmitted to the brain during impact. Axonal injury leads to brain network dysfunction, significantly contributing to cognitive and functional impairments frequently observed in TBI survivors. Diffuse axonal injury (DAI is a clinical entity suggested by impaired level of consciousness and coma on clinical examination and characterized by widespread injury to the hemispheric white matter tracts, the corpus callosum and the brain stem. The clinical course of DAI is commonly unpredictable and it remains a challenging entity with limited therapeutic options, to date. Although axonal integrity may be disrupted at impact, the majority of axonal pathology evolves over time, resulting from delayed activation of complex intracellular biochemical cascades. Activation of these secondary biochemical pathways may lead to axonal transection, named secondary axotomy, and be responsible for the clinical decline of DAI patients. Advances in the neurocritical care of TBI patients have been achieved by refinements in multimodality monitoring for prevention and early detection of secondary injury factors, which can be applied also to DAI. There is an emerging role for biomarkers in blood, cerebrospinal fluid, and interstitial fluid using microdialysis in the evaluation of axonal injury in TBI. These biomarker studies have assessed various axonal and neuroglial markers as well as inflammatory mediators, such as cytokines and chemokines. Moreover, modern neuroimaging can detect subtle or overt DAI/white matter changes in diffuse TBI patients across all injury severities using magnetic resonance spectroscopy, diffusion tensor imaging, and positron emission tomography. Importantly, serial neuroimaging studies provide evidence for evolving axonal injury. Since axonal injury may be a key

  18. Axonal degeneration stimulates the formation of NG2+ cells and oligodendrocytes in the mouse

    DEFF Research Database (Denmark)

    Nielsen, Helle Hvilsted; Ladeby, Rune; Drøjdahl, Nina


    the response of the NG2+ cells to the different components of demyelinating pathology, we investigated the response of adult NG2+ cells to axonal degeneration in the absence of primary myelin or oligodendrocyte pathology. Axonal degeneration was induced in the hippocampal dentate gyrus of adult mice...... by transection of the entorhino-dentate perforant path projection. The acutely induced degeneration of axons and terminals resulted in a prompt response of NG2+ cells, consisting of morphological transformation, cellular proliferation, and upregulation of NG2 expression days 2-3 after surgery. This was followed...

  19. Akt2-Dependent Phosphorylation of Radixin in Regulation of Mrp-2 Trafficking in WIF-B Cells. (United States)

    Suda, Jo; Rockey, Don C; Karvar, Serhan


    The dominant ezrin/radixin/moesin protein in hepatocytes is radixin, which plays an important role in mediating the binding of F-actin to the plasma membrane after a conformational activation by phosphorylation at Thr564. Here we have investigated the importance of Akt-mediated radixin Thr564 phosphorylation on Mrp-2 distribution and function in WIF-B cells. Mrp-2 is an adenosine triphosphate (ATP)-binding cassette transporter that plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, organic anions, and drug metabolites such as glucuronides. Akt1 and Akt2 expression were manipulated using dominant active and negative constructs as well as Akt1 and Akt2 siRNA. Cellular distribution of radixin and Mrp-2 was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate, which is a substrate of the Mrp-2 and is actively transported in canalicular lumina, was used to measure Mrp-2 function. Radixin phosphorylation was significantly increased in wild-type and dominant active Akt2 transfected cells. Furthermore, radixin and Mrp-2 were localized at the canalicular membrane, similar to control cells. In contrast, overexpression of dominant negative Akt2, siRNA knockdown of Akt2 and a specific Akt inhibitor prevented radixin phosphorylation and led to alteration of normal radixin and Mrp-2 localization; inhibition of Akt2, but not Akt1 function led to radixin localization to the cytoplasmic space. In addition, dominant negative and Akt2 knockdown led to a dramatically impaired hepatocyte secretory response, while wild-type and dominant active Akt2 transfected cells exhibited increased 5-chloromethylfluorescein diacetate excretion. In contrast to Akt2, Akt1 was not associated with radixin phosphorylation. These studies, therefore, identify Akt2 as a critical kinase that regulates radixin phosphorylation and leads to Mrp-2 translocation and

  20. An Atypical SCF-like Ubiquitin Ligase Complex Promotes Wallerian Degeneration through Regulation of Axonal Nmnat2

    Directory of Open Access Journals (Sweden)

    Yuya Yamagishi


    Full Text Available Axon degeneration is a tightly regulated, self-destructive program that is a critical feature of many neurodegenerative diseases, but the molecular mechanisms regulating this program remain poorly understood. Here, we identify S-phase kinase-associated protein 1A (Skp1a, a core component of a Skp/Cullin/F-box (SCF-type E3 ubiquitin ligase complex, as a critical regulator of axon degeneration after injury in mammalian neurons. Depletion of Skp1a prolongs survival of injured axons in vitro and in the optic nerve in vivo. We demonstrate that Skp1a regulates the protein level of the nicotinamide adenine dinucleotide (NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2 in axons. Loss of axonal Nmnat2 contributes to a local ATP deficit that triggers axon degeneration. Knockdown of Skp1a elevates basal levels of axonal Nmnat2, thereby delaying axon degeneration through prolonged maintenance of axonal ATP. Consistent with Skp1a functioning through regulation of Nmnat2, Skp1a knockdown fails to protect axons from Nmnat2 knockdown. These results illuminate the molecular mechanism underlying Skp1a-dependent axonal destruction.

  1. Effects of 1,2,4-Trichlorobenzene and Mercury Ion Stress on Ca2+ Fluxion and Protein Phosphorylation in Rice

    Directory of Open Access Journals (Sweden)

    Cai-lin GE


    Full Text Available The effects of 5 mg/L 1,2,4-trichlorobenzene (TCB and 0.1 mmol/L mercury ion (Hg2+ stresses on Ca2+ fluxion and protein phosphorylation in rice seedlings were investigated by isotope exchange kinetics and in vitro phosphorylation assay. The Ca2+ absorption in rice leaves and Ca2+ transportation from roots to leaves were promoted significantly in response to Hg2+ and TCB treatments for 4-48 h. The Ca2+ absorption peaks presented in the leaves when the rice seedlings were exposed to Hg2+ for 8-12 h or to TCB for 12-24 h. Several Ca2+ absorption peaks presented in the roots during rice seedlings being exposed to Hg2+ and TCB, and the first Ca2+ absorption peak was at 8 h after being exposed to Hg2+ and TCB. The result of isotope exchange kinetic analysis confirmed that short-term (8 h Hg2+ and TCB stresses caused Ca2+ channels or pumps located on plasmalemma to open transiently. The phosphorylation assay showed that short-term TCB stress enhanced protein phosphorylation in rice roots (TCB treatment for 4-8 h and leaves (TCB treatment for 4-24 h, and short-term (4-8 h Hg2+ stress also enhanced protein phosphorylation in rice leaves. The enhancement of protein phosphorylation in both roots and leaves corresponded with the first Ca2+ absorption peak, which confirmed that the enhancement of protein phosphorylation caused by TCB or Hg2+ stress might be partly triggered by the increases of cytosolic calcium. TCB treatment over 12 h inhibited protein phosphorylation in rice roots, which might be partly due to that TCB stress suppressed the protein kinase activity. Whereas, Hg2+ treatment inhibited protein phosphorylation in rice roots, and Hg2+ treatment over 12 h inhibited protein phosphorylation in rice leaves. This might be attributed to that not only the protein kinase activity, but also the expressions of phosphorylation proteins were restrained by Hg2+ stress.

  2. Guidance of Drosophila Mushroom Body Axons Depends upon DRL-Wnt Receptor Cleavage in the Brain Dorsomedial Lineage Precursors

    Directory of Open Access Journals (Sweden)

    Elodie Reynaud


    Full Text Available In vivo axon pathfinding mechanisms in the neuron-dense brain remain relatively poorly characterized. We study the Drosophila mushroom body (MB axons, whose α and β branches connect to different brain areas. We show that the Ryk family WNT5 receptor, DRL (derailed, which is expressed in the dorsomedial lineages, brain structure precursors adjacent to the MBs, is required for MB α branch axon guidance. DRL acts to capture and present WNT5 to MB axons rather than transduce a WNT5 signal. DRL’s ectodomain must be cleaved and shed to guide α axons. DRL-2, another Ryk, is expressed within MB axons and functions as a repulsive WNT5 signaling receptor. Finally, our biochemical data support the existence of a ternary complex composed of the cleaved DRL ectodomain, WNT5, and DRL-2. Thus, the interaction of MB-extrinsic and -intrinsic Ryks via their common ligand acts to guide MB α axons.

  3. Activating PER repressor through a DBT-directed phosphorylation switch.

    Directory of Open Access Journals (Sweden)

    Saul Kivimäe


    Full Text Available Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT, a Drosophila ortholog of human casein kinase I (CKIepsilon/delta. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER. DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The per(S mutation, which is associated with short-period (19-h circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function.

  4. Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides (United States)

    Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.


    In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

  5. Can you hear me now? Regulating transcriptional activators by phosphorylation. (United States)

    Gardner, Kevin H; Montminy, Marc


    Extracellular signals often modulate the expression of specific genetic programs by triggering the phosphorylation of relevant transcription factors (TFs). Phosphorylation in turn regulates such TFs by altering their cellular localization, DNA binding affinity, or transcriptional activity. Structural approaches have revealed how phosphorylation turns some TFs on or off; but less is known about how phosphorylation regulates other transcription factors in a graded manner that depends on signal intensity. A recent paper by Graves and colleagues reveals how a group of phosphorylation sites in Ets-1 regulates its DNA binding activity. Their studies provide new insight into the importance of multisite phosphorylation for the graded regulation of transcription and highlight the involvement of allosteric mechanisms in this process.

  6. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.


    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

  7. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen


    . In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomic study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins....... Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes....... in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates...

  8. Kinase-specific prediction of protein phosphorylation sites

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Blom, Nikolaj


    As extensive mass spectrometry-based mapping of the phosphoproteome progresses, computational analysis of phosphorylation-dependent signaling becomes increasingly important. The linear sequence motifs that surround phosphorylated residues have successfully been used to characterize kinase......-substrate specificity. Here, we briefly describe the available resources for predicting kinase-specific phosphorylation from sequence properties. We address the strengths and weaknesses of these resources, which are based on methods ranging from simple consensus patterns to more advanced machine-learning algorithms...

  9. The in vivo phosphorylation sites of rat brain dynamin I

    DEFF Research Database (Denmark)

    Graham, Mark E; Anggono, Victor; Bache, Nicolai


    Dynamin I (dynI) is phosphorylated in synaptosomes at Ser(774) and Ser(778) by cyclin-dependent kinase 5 to regulate recruitment of syndapin I for synaptic vesicle endocytosis, and in PC12 cells on Ser(857). Hierarchical phosphorylation of Ser(774) precedes phosphorylation of Ser(778). In contrast......, Thr(780) phosphorylation by cdk5 has been reported as the sole site (Tomizawa, K., Sunada, S., Lu, Y. F., Oda, Y., Kinuta, M., Ohshima, T., Saito, T., Wei, F. Y., Matsushita, M., Li, S. T., Tsutsui, K., Hisanaga, S. I., Mikoshiba, K., Takei, K., and Matsui, H. (2003) J. Cell Biol. 163, 813...

  10. Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

    DEFF Research Database (Denmark)

    Caspersen, M.B.; Qiu, J.-L.; Zhang, X.


    The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...

  11. Altered phosphorylation of rhodopsin in retinal dystrophic Irish Setters

    International Nuclear Information System (INIS)

    Cunnick, J.; Takemoto, D.J.; Takemoto, L.J.


    The carboxyl-terminus of rhodopsin in retinal dystrophic (rd) Irish Setters is altered near a possible phosphorylation site. To determine if this alteration affects ATP-mediated phosphorylation they compared the phosphorylation of rhodopsin from rd affected Irish Setters and normal unaffected dogs. Retinas from 8-week-old Irish Setters were phosphorylated with γ- 32 P-ATP and separated on SDS-PAGE. Compared to unaffected normal retinas, equalized for rhodopsin content, phosphorylation of rd rhodopsin was drastically reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Inhibition also occurred when bovine retinas were mixed with rd retinas. The rd-mediated inhibition of phosphorylation was prevented by including 1mM NaF in the reaction mixture. Likewise, 1mM NaF restored phosphorylation of rd rhodopsin to normal levels. Phosphopeptide maps of rd and normal rhodopsin were identical and indicated 5 phosphopeptides present in each. Results suggest that one cause of the depressed rd rhodopsin phosphorylation is an increased phosphatase activity

  12. Islet Coordinately Regulates Motor Axon Guidance and Dendrite Targeting through the Frazzled/DCC Receptor

    Directory of Open Access Journals (Sweden)

    Celine Santiago


    Full Text Available Motor neuron axon targeting in the periphery is correlated with the positions of motor neuron inputs in the CNS, but how these processes are coordinated to form a myotopic map remains poorly understood. We show that the LIM homeodomain factor Islet (Isl controls targeting of both axons and dendrites in Drosophila motor neurons through regulation of the Frazzled (Fra/DCC receptor. Isl is required for fra expression in ventrally projecting motor neurons, and isl and fra mutants have similar axon guidance defects. Single-cell labeling indicates that isl and fra are also required for dendrite targeting in a subset of motor neurons. Finally, overexpression of Fra rescues axon and dendrite targeting defects in isl mutants. These results indicate that Fra acts downstream of Isl in both the periphery and the CNS, demonstrating how a single regulatory relationship is used in multiple cellular compartments to coordinate neural circuit wiring.

  13. Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury* (United States)

    van Niekerk, Erna A.; Tuszynski, Mark H.; Lu, Paul; Dulin, Jennifer N.


    Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system. PMID:26695766

  14. Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury. (United States)

    van Niekerk, Erna A; Tuszynski, Mark H; Lu, Paul; Dulin, Jennifer N


    Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Vertebrate Fidgetin Restrains Axonal Growth by Severing Labile Domains of Microtubules

    Directory of Open Access Journals (Sweden)

    Lanfranco Leo


    Full Text Available Individual microtubules (MTs in the axon consist of a stable domain that is highly acetylated and a labile domain that is not. Traditional MT-severing proteins preferentially cut the MT in the stable domain. In Drosophila, fidgetin behaves in this fashion, with targeted knockdown resulting in neurons with a higher fraction of acetylated (stable MT mass in their axons. Conversely, in a fidgetin knockout mouse, the fraction of MT mass that is acetylated is lower than in the control animal. When fidgetin is depleted from cultured rodent neurons, there is a 62% increase in axonal MT mass, all of which is labile. Concomitantly, there are more minor processes and a longer axon. Together with experimental data showing that vertebrate fidgetin targets unacetylated tubulin, these results indicate that vertebrate fidgetin (unlike its fly ortholog regulates neuronal development by tamping back the expansion of the labile domains of MTs.

  16. Lithium Enhances Axonal Regeneration in Peripheral Nerve by Inhibiting Glycogen Synthase Kinase 3β Activation

    Directory of Open Access Journals (Sweden)

    Huanxing Su


    Full Text Available Brachial plexus injury often involves traumatic root avulsion resulting in permanent paralysis of the innervated muscles. The lack of sufficient regeneration from spinal motoneurons to the peripheral nerve (PN is considered to be one of the major causes of the unsatisfactory outcome of various surgical interventions for repair of the devastating injury. The present study was undertaken to investigate potential inhibitory signals which influence axonal regeneration after root avulsion injury. The results of the study showed that root avulsion triggered GSK-3β activation in the injured motoneurons and remaining axons in the ventral funiculus. Systemic application of a clinical dose of lithium suppressed activated GSK-3β in the lesioned spinal cord to the normal level and induced extensive axonal regeneration into replanted ventral roots. Our study suggests that GSK-3β activity is involved in negative regulation for axonal elongation and regeneration and lithium, the specific GSK-3β inhibitor, enhances motoneuron regeneration from CNS to PNS.

  17. Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling

    DEFF Research Database (Denmark)

    Cheng, Jin; Sahani, Sadhna; Hausrat, Torben Johann


    Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in...

  18. LRP1 controls cPLA2 phosphorylation, ABCA1 expression and cellular cholesterol export.

    Directory of Open Access Journals (Sweden)

    Li Zhou


    Full Text Available ATP-binding cassette transporter A1 mediates apolipoprotein AI-dependent efflux of cholesterol and thereby removes cholesterol from peripheral tissues. ABCA1 expression is tightly regulated and deficiency of this cholesterol transporter results in cholesterol accumulation within cells. Low-density lipoprotein receptor-related protein 1 (LRP1 participates in lipid metabolism and energy homeostasis by endocytosis of apolipoprotein E-containing lipoproteins and modulation of cellular proliferation signals.In the present study, we demonstrate a new role for LRP1 in reverse cholesterol transport. Absence of LRP1 expression results in increased PDGFRbeta signaling and sequential activation of the mitogen-activated protein kinase signaling pathway, which increases phosphorylation of cytosolic phospholipase A(2 (cPLA(2. Phosphorylated and activated cPLA(2 releases arachidonic acid from the phospholipid pool. Overproduction of arachidonic acid suppresses the activation of LXR/RXR heterodimers bound to the promoter of LXR regulated genes such as ABCA1, resulting in greatly reduced ABCA1 expression.LRP1 regulates LXR-mediated gene transcription and participates in reverse cholesterol transport by controlling cPLA(2 activation and ABCA1 expression. LRP1 thus functions as a physiological integrator of cellular lipid homeostasis with signals that regulate cellular proliferation and vascular wall integrity.

  19. Tormenta simpática paroxística siguiendo a injuria Axonal difusa Paroxysmal sympathetic storm after diffuse axonal head injury


    Pablo Young; Barbara C. Finn; Debora Pellegrini; Elias D. Soloaga; Julio E. Bruetman


    El término tormenta simpática paroxística se utiliza como sinónimo de alteraciones episódicas de la temperatura corporal, la presión arterial, la frecuencia respiratoria y cardíaca, el tamaño pupilar y el nivel de conciencia, que coinciden con hiperhidrosis, salivación excesiva y postura extensora. Esto siempre en el contexto de una injuria axonal difusa grave que sigue a un traumatismo encéfalo-craneano (TEC) grave. Presentamos dos pacientes jóvenes con injuria axonal difusa secundaria a TEC...

  20. Chondroitin sulfate proteoglycans negatively regulate the positioning of mitochondria and endoplasmic reticulum to distal axons. (United States)

    Sainath, Rajiv; Armijo-Weingart, Lorena; Ketscheck, Andrea; Xu, Zhuxuan; Li, Shuxin; Gallo, Gianluca


    Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter-linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150 Glu dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1351-1370, 2017. © 2017 Wiley Periodicals, Inc.

  1. Selective axonal growth of embryonic hippocampal neurons according to topographic features of various sizes and shapes

    Directory of Open Access Journals (Sweden)

    Christine E Schmidt


    Full Text Available David Y Fozdar1*, Jae Y Lee2*, Christine E Schmidt2–6, Shaochen Chen1,3–5,7,1Departments of Mechanical Engineering, 2Chemical Engineering, 3Biomedical Engineering; 4Center for Nano Molecular Science and Technology; 5Texas Materials Institute; 6Institute of Neuroscience; 7Microelectronics Research Center, The University of Texas at Austin, Austin, TX, USA *Contributed equally to this workPurpose: Understanding how surface features influence the establishment and outgrowth of the axon of developing neurons at the single cell level may aid in designing implantable scaffolds for the regeneration of damaged nerves. Past studies have shown that micropatterned ridge-groove structures not only instigate axon polarization, alignment, and extension, but are also preferred over smooth surfaces and even neurotrophic ligands.Methods: Here, we performed axonal-outgrowth competition assays using a proprietary four-quadrant topography grid to determine the capacity of various micropatterned topographies to act as stimuli sequestering axon extension. Each topography in the grid consisted of an array of microscale (approximately 2 µm or submicroscale (approximately 300 nm holes or lines with variable dimensions. Individual rat embryonic hippocampal cells were positioned either between two juxtaposing topographies or at the borders of individual topographies juxtaposing unpatterned smooth surface, cultured for 24 hours, and analyzed with respect to axonal selection using conventional imaging techniques.Results: Topography was found to influence axon formation and extension relative to smooth surface, and the distance of neurons relative to topography was found to impact whether the topography could serve as an effective cue. Neurons were also found to prefer submicroscale over microscale features and holes over lines for a given feature size.Conclusion: The results suggest that implementing physical cues of various shapes and sizes on nerve guidance conduits

  2. Developmental plasticity of ascending spinal axons studies using the North American opossum, Didelphis virginiana. (United States)

    Terman, J R; Wang, X M; Martin, G F


    The objectives of the present study were to determine if axons of all ascending tracts grow through the lesion after transection of the thoracic spinal cord during development in the North American opossum, and if so, whether they reach regions of the brain they normally innervate. Opossum pups were subjected to transection of the mid-thoracic cord at PD5, PD8, PD12, PD20, or PD26 and injections of Fast Blue (FB) into the lower thoracic or upper lumbar cord 30-40 days or 6 months later. In the PD5 transected cases, labeled axons were present in all of the supraspinal areas labeled by comparable injections in unlesioned, age-matched controls. In the experimental cases, however, labeled axons appeared to be fewer in number and in some areas more restricted in location than in the controls. When lesions were made at PD8, labeled axons were present in the brain of animals allowed to survive 30-40 days prior to FB injections but they were not observed in those allowed to survive 6 months. When lesions were made at PD12 or later, labeled axons were never found rostral to the lesion. It appears, therefore, that axons of all ascending spinal pathways grow though the lesion after transection of the thoracic cord in developing opossums and that they innervate appropriate areas of the brain. Interestingly, the critical period for such growth is shorter than that for most descending axons, suggesting that factors which influence loss of developmental plasticity are not the same for all axons.

  3. Activation of ganglion cells and axon bundles using epiretinal electrical stimulation. (United States)

    Grosberg, Lauren E; Ganesan, Karthik; Goetz, Georges A; Madugula, Sasidhar S; Bhaskhar, Nandita; Fan, Victoria; Li, Peter; Hottowy, Pawel; Dabrowski, Wladyslaw; Sher, Alexander; Litke, Alan M; Mitra, Subhasish; Chichilnisky, E J


    Epiretinal prostheses for treating blindness activate axon bundles, causing large, arc-shaped visual percepts that limit the quality of artificial vision. Improving the function of epiretinal prostheses therefore requires understanding and avoiding axon bundle activation. This study introduces a method to detect axon bundle activation on the basis of its electrical signature and uses the method to test whether epiretinal stimulation can directly elicit spikes in individual retinal ganglion cells without activating nearby axon bundles. Combined electrical stimulation and recording from isolated primate retina were performed using a custom multielectrode system (512 electrodes, 10-μm diameter, 60-μm pitch). Axon bundle signals were identified by their bidirectional propagation, speed, and increasing amplitude as a function of stimulation current. The threshold for bundle activation varied across electrodes and retinas, and was in the same range as the threshold for activating retinal ganglion cells near their somas. In the peripheral retina, 45% of electrodes that activated individual ganglion cells (17% of all electrodes) did so without activating bundles. This permitted selective activation of 21% of recorded ganglion cells (7% of expected ganglion cells) over the array. In one recording in the central retina, 75% of electrodes that activated individual ganglion cells (16% of all electrodes) did so without activating bundles. The ability to selectively activate a subset of retinal ganglion cells without axon bundles suggests a possible novel architecture for future epiretinal prostheses. NEW & NOTEWORTHY Large-scale multielectrode recording and stimulation were used to test how selectively retinal ganglion cells can be electrically activated without activating axon bundles. A novel method was developed to identify axon activation on the basis of its unique electrical signature and was used to find that a subset of ganglion cells can be activated at single

  4. The Impact of Motor Axon Misdirection and Attrition on Behavioral Deficit Following Experimental Nerve Injuries (United States)

    Alant, Jacob Daniel de Villiers; Senjaya, Ferry; Ivanovic, Aleksandra; Forden, Joanne; Shakhbazau, Antos; Midha, Rajiv


    Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (pinjuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize

  5. Dysfunction of axonal membrane conductances in adolescents and young adults with spinal muscular atrophy


    Farrar, Michelle A.; Vucic, Steve; Lin, Cindy S.-Y.; Park, Susanna B.; Johnston, Heather M.; du Sart, Desir?e; Bostock, Hugh; Kiernan, Matthew C.


    Spinal muscular atrophy is distinct among neurodegenerative conditions of the motor neuron, with onset in developing and maturing patients. Furthermore, the rate of degeneration appears to slow over time, at least in the milder forms. To investigate disease pathophysiology and potential adaptations, the present study utilized axonal excitability studies to provide insights into axonal biophysical properties and explored correlation with clinical severity. Multiple excitability indices (stimul...

  6. Independent signaling by Drosophila insulin receptor for axon guidance and growth

    Directory of Open Access Journals (Sweden)

    Caroline Rita Li


    Full Text Available The Drosophila insulin receptor (DInR regulates a diverse array of biological processes including growth, axon guidance, and sugar homeostasis. Growth regulation by DInR is mediated by Chico, the Drosophila homolog of vertebrate insulin-receptor-substrate proteins IRS1-4. In contrast, DInR regulation of photoreceptor axon guidance in the developing visual system is mediated by the SH2-SH3 domain adaptor protein Dreadlocks (Dock. In vitro studies by others identified five NPXY motifs, one in the juxtamembrane region and four in the signaling C-terminal tail (C-tail, important for interaction with Chico. Here we used yeast two-hybrid assays to identify regions in the DInR C-tail that interact with Dock. These Dock-binding sites were in separate portions of the C-tail from the previously identified Chico-binding sites. To test whether these sites are required for growth or axon guidance in whole animals, a panel of DInR proteins, in which the putative Chico and Dock interaction sites had been mutated individually or in combination, were tested for their ability to rescue viability, growth, and axon guidance defects of dinr mutant flies. Sites required for viability were identified. Unexpectedly, mutation of both putative Dock binding sites, either individually or in combination, did not lead to defects in photoreceptor axon guidance. Thus, either sites also required for viability are necessary for DInR function in axon guidance and/or there is redundancy built into the DInR/Dock interaction such that Dock is able to interact with multiple regions of DInR. We also found that simultaneous mutation of all 5 NPXY motifs implicated in Chico interaction drastically decreased growth in both male and female adult flies. Mutation of these 5 NPXY motifs did not affect photoreceptor axon guidance, showing that different sites within DInR control growth and axon guidance.

  7. Hydrogels as scaffolds and delivery systems to enhance axonal regeneration after injuries

    Directory of Open Access Journals (Sweden)

    Oscar A. Carballo-Molina


    Full Text Available Damage caused to neural tissue by disease or injury frequently produces a discontinuity in the nervous system. Such damage generates diverse alterations that are commonly permanent, due to the limited regeneration capacity of the adult nervous system, particularly the Central Nervous System (CNS. The cellular reaction to noxious stimulus leads to several events such as the formation of glial and fibrous scars, which inhibit axonal regeneration in both the CNS and the Peripheral Nervous System (PNS. Although in the PNS there is some degree of nerve regeneration, it is common that the growing axons reinnervate incorrect areas, causing mismatches. Providing a permissive substrate for axonal regeneration in combination with delivery systems for the release of molecules, which enhances axonal growth, could increase regeneration and the recovery of functions in the CNS or the PNS. Currently, there are no effective vehicles to supply growth factors or cells to the damaged/diseased nervous system. Hydrogels are polymers that are biodegradable, biocompatible and have the capacity to deliver a large range of molecules in situ. The inclusion of cultured neural cells into hydrogels forming three-dimensional structures allows the formation of synapses and neuronal survival. There is also evidence showing that hydrogels constitute an amenable substrate for axonal growth of endogenous or grafted cells, overcoming the presence of axonal regeneration inhibitory molecules, in both the central and peripheral nervous systems. Recent experiments suggest that hydrogels can carry and deliver several proteins relevant for improving neuronal survival and axonal growth. Although the use of hydrogels is appealing, its effectiveness is still a matter of discussion, and more results are needed to achieve consistent recovery using different parameters. This review also discusses areas of opportunity where hydrogels can be applied, in order to promote axonal regeneration of




    The effects of detergents on squid giant axon action and resting potentials as well as membrane conductances in the voltage clamp have been studied. Anionic detergents (sodium lauryl sulfate, 0.1 to 1.0 mM; dimethyl benzene sulfonate, 1 to 20 mM, pH 7.6) cause a temporary increase and a later decrease of action potential height and the value of the resting potential. Cationic detergent (cetyl trimethyl ammonium chloride, 6 x 10(-5)M or more, pH 7.6) generally brings about immediate and irreversible decreases in the action and resting potentials. Non-ionic detergent (tween 80, 0.1 M, pH 7.6) causes a slight reversible reduction of action potential height without affecting the value of the resting potential. Both anionic and cationic detergents generally decrease the sodium and potassium conductances irreversibly. The effect of non-ionic detergent is to decrease the sodium conductance reversibly, leaving the potassium conductance almost unchanged.

  9. Axon ensheathment and metabolic supply by glial cells in Drosophila. (United States)

    Schirmeier, Stefanie; Matzat, Till; Klämbt, Christian


    Neuronal function requires constant working conditions and a well-balanced supply of ions and metabolites. The metabolic homeostasis in the nervous system crucially depends on the presence of glial cells, which nurture and isolate neuronal cells. Here we review recent findings on how these tasks are performed by glial cells in the genetically amenable model organism Drosophila melanogaster. Despite the small size of its nervous system, which would allow diffusion of metabolites, a surprising division of labor between glial cells and neurons is evident. Glial cells are glycolytically active and transfer lactate and alanine to neurons. Neurons in turn do not require glycolysis but can use the glially provided compounds for their energy homeostasis. Besides feeding neurons, glial cells also insulate neuronal axons in a way similar to Remak fibers in the mammalian nervous system. The molecular mechanisms orchestrating this insulation require neuregulin signaling and resemble the mechanisms controlling glial differentiation in mammals surprisingly well. We hypothesize that metabolic cross talk and insulation of neurons by glial cells emerged early during evolution as two closely interlinked features in the nervous system. This article is part of a Special Issue entitled SI: Myelin Evolution. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Mechanisms of neurodegeneration and axonal dysfunction in multiple sclerosis. (United States)

    Friese, Manuel A; Schattling, Benjamin; Fugger, Lars


    Multiple sclerosis (MS) is the most frequent chronic inflammatory disease of the CNS, and imposes major burdens on young lives. Great progress has been made in understanding and moderating the acute inflammatory components of MS, but the pathophysiological mechanisms of the concomitant neurodegeneration--which causes irreversible disability--are still not understood. Chronic inflammatory processes that continuously disturb neuroaxonal homeostasis drive neurodegeneration, so the clinical outcome probably depends on the balance of stressor load (inflammation) and any remaining capacity for neuronal self-protection. Hence, suita