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Sample records for axonal transport characteristics

  1. Cargo distributions differentiate pathological axonal transport impairments.

    PubMed

    Mitchell, Cassie S; Lee, Robert H

    2012-05-07

    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.

  2. Cargo distributions differentiate pathological axonal transport impairments

    PubMed Central

    Mitchell, Cassie S.; Lee, Robert H.; Coulter, Wallace H.

    2012-01-01

    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. PMID:22285784

  3. CNS axons globally increase axonal transport after peripheral conditioning.

    PubMed

    Mar, Fernando M; Simões, Anabel R; Leite, Sérgio; Morgado, Marlene M; Santos, Telma E; Rodrigo, Inês S; Teixeira, Carla A; Misgeld, Thomas; Sousa, Mónica M

    2014-04-23

    Despite the inability of CNS axons to regenerate, an increased regenerative capacity can be elicited following conditioning lesion to the peripheral branch of dorsal root ganglia neurons (DRGs). By in vivo radiolabeling of rat DRGs, coupled to mass spectrometry and kinesin immunoprecipitation of spinal cord extracts, we determined that the anterograde transport of cytoskeleton components, metabolic enzymes and axonal regeneration enhancers, was increased in the central branch of DRGs following a peripheral conditioning lesion. Axonal transport of mitochondria was also increased in the central branch of Thy1-MitoCFP mice following a peripheral injury. This effect was generalized and included augmented transport of lysosomes and synaptophysin- and APP-carrying vesicles. Changes in axonal transport were only elicited by a peripheral lesion and not by spinal cord injury. In mice, elevated levels of motors and of polyglutamylated and tyrosinated tubulin were present following a peripheral lesion and can explain the increase in axonal transport induced by conditioning. In summary, our work shows that a peripheral injury induces a global increase in axonal transport that is not restricted to the peripheral branch, and that, by extending to the central branch, allows a rapid and sustained support of regenerating central axons.

  4. The axonal transport of mitochondria

    PubMed Central

    Saxton, William M.; Hollenbeck, Peter J.

    2012-01-01

    Vigorous transport of cytoplasmic components along axons over substantial distances is crucial for the maintenance of neuron structure and function. The transport of mitochondria, which serves to distribute mitochondrial functions in a dynamic and non-uniform fashion, has attracted special interest in recent years following the discovery of functional connections among microtubules, motor proteins and mitochondria, and their influences on neurodegenerative diseases. Although the motor proteins that drive mitochondrial movement are now well characterized, the mechanisms by which anterograde and retrograde movement are coordinated with one another and with stationary axonal mitochondria are not yet understood. In this Commentary, we review why mitochondria move and how they move, focusing particularly on recent studies of transport regulation, which implicate control of motor activity by specific cell-signaling pathways, regulation of motor access to transport tracks and static microtubule–mitochondrion linkers. A detailed mechanism for modulating anterograde mitochondrial transport has been identified that involves Miro, a mitochondrial Ca2+-binding GTPase, which with associated proteins, can bind and control kinesin-1. Elements of the Miro complex also have important roles in mitochondrial fission–fusion dynamics, highlighting questions about the interdependence of biogenesis, transport, dynamics, maintenance and degradation. PMID:22619228

  5. Fast axonal transport in early experimental disc edema.

    PubMed

    Radius, R L; Anderson, D R

    1980-02-01

    Previous work has documented impairment of slow axonal transport in papilledema, but the abnormalities in rapid transport were less certain. Therefore fast axonal transport was studied in 19 primate eyes subjected to ocular hypotony for 6 to 72 hr following surgical fistulization of the anterior chamber. Mild, irregular alterations in fast axonal transport were detected only after nerve head swelling was apparent. These changes in fast transport mechanisms in cases of nerve head edema occur after, and may be secondary to, impaired slow axoplasmic flow and the resultant axonal swelling. Furthermore, since prolonged complete interruption of axonal transport is theoretically inconsistent with the continued normal neuron function characteristic of papilledema and, moreover, since previous data shows a "slowdown" rather than complete blockade of axonal transport in papilledema, it is likely that in eyes with papilledema there does not exist a complete flock of axonal transport. Therefore we hypothesize that the swelling results when slow axoplasmic flow is locally slowed down but not totally stopped, with the axon distention producing secondary mild, irregular changes in fast axonal transport.

  6. Dynamics of Mitochondrial Transport in Axons

    PubMed Central

    Niescier, Robert F.; Kwak, Sang Kyu; Joo, Se Hun; Chang, Karen T.; Min, Kyung-Tai

    2016-01-01

    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. PMID:27242435

  7. Methodological advances in imaging intravital axonal transport.

    PubMed

    Sleigh, James N; Vagnoni, Alessio; Twelvetrees, Alison E; Schiavo, Giampietro

    2017-01-01

    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.

  8. Methodological advances in imaging intravital axonal transport

    PubMed Central

    Sleigh, James N.; Vagnoni, Alessio; Twelvetrees, Alison E.; Schiavo, Giampietro

    2017-01-01

    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. PMID:28344778

  9. Morphology of axonal transport abnormalities in primate eyes.

    PubMed

    Radius, R L; Anderson, D R

    1981-11-01

    The ultrastructure of the retina and optic nerve head was studied in primate eyes after central retinal artery occlusion. Within 2 hours of the vascular occlusion the inner retinal layers undergo watery (isosmotic) swelling. This watery swelling of axons and astroglia extends into the nerve head as far back as the anterior boundary of the scleral lamina cribrosa. The swelling is increased 4 hours after the occlusion, and by 24 hours disintegration has occurred. At the optic nerve head mitochondria and vesicles of smooth endoplasmic reticulum begin to accumulate within 2 hours. The accumulation increases at 4 hours and persists to 24 hours. The watery swelling seems characteristic of ischaemic axons. Membranous organelles accumulate at the boundary of an ischaemic zone when material carried by axonal transport is brought via the healthy axon segment to the boundary, but they cannot proceed further into the ischaemic zone. Such accumulation is typical of locations where rapid orthograde axonal transport or retrograde axonal transport is blocked. In contrast, when slow axonal flow is impaired, the swelling is characterised by an excess of cytoplasmic gel without a marked accumulation of organelles. Rapid orthograde transport and retrograde transport seem to be closely related to one another, while slow axoplasmic flow seems fundamentally different. From morphological findings we suspect that, in experimental glaucoma, intraocular pressure first affects the intracellular physiological process of rapid orthograde and retrograde axonal transport. Watery swelling may not occur unless the ischaemic injury to cell metabolism is more advanced. In contrast, in experimental papilloedema, the swelling results predominantly from impaired slow axoplasmic flow.

  10. Morphology of axonal transport abnormalities in primate eyes.

    PubMed Central

    Radius, R L; Anderson, D R

    1981-01-01

    The ultrastructure of the retina and optic nerve head was studied in primate eyes after central retinal artery occlusion. Within 2 hours of the vascular occlusion the inner retinal layers undergo watery (isosmotic) swelling. This watery swelling of axons and astroglia extends into the nerve head as far back as the anterior boundary of the scleral lamina cribrosa. The swelling is increased 4 hours after the occlusion, and by 24 hours disintegration has occurred. At the optic nerve head mitochondria and vesicles of smooth endoplasmic reticulum begin to accumulate within 2 hours. The accumulation increases at 4 hours and persists to 24 hours. The watery swelling seems characteristic of ischaemic axons. Membranous organelles accumulate at the boundary of an ischaemic zone when material carried by axonal transport is brought via the healthy axon segment to the boundary, but they cannot proceed further into the ischaemic zone. Such accumulation is typical of locations where rapid orthograde axonal transport or retrograde axonal transport is blocked. In contrast, when slow axonal flow is impaired, the swelling is characterised by an excess of cytoplasmic gel without a marked accumulation of organelles. Rapid orthograde transport and retrograde transport seem to be closely related to one another, while slow axoplasmic flow seems fundamentally different. From morphological findings we suspect that, in experimental glaucoma, intraocular pressure first affects the intracellular physiological process of rapid orthograde and retrograde axonal transport. Watery swelling may not occur unless the ischaemic injury to cell metabolism is more advanced. In contrast, in experimental papilloedema, the swelling results predominantly from impaired slow axoplasmic flow. Images PMID:6173060

  11. Axonal transport disruption in peripheral nerve disease

    PubMed Central

    Lloyd, Thomas E.

    2015-01-01

    Many neurodegenerative diseases and neuropathies have been proposed to be caused by a disruption of axonal transport. However, the mechanisms whereby impaired transport causes disease remain unclear. Proposed mechanisms include impairment in delivery of organelles such as mitochondria, defective retrograde neurotrophic signaling, and disruption of the synaptic vesicle cycle within the synaptic terminal. Simple model organisms such as the fruitfly, Drosophila melanogaster, allow live imaging of axonal transport to be combined with high-throughput genetic screens and are providing insights into the pathophysiology of peripheral nerve diseases. PMID:23279432

  12. AXONAL TRANSPORT: CARGO-SPECIFIC MECHANISMS OF MOTILITY AND REGULATION

    PubMed Central

    Maday, Sandra; Twelvetrees, Alison E.; Moughamian, Armen J.; Holzbaur, Erika L. F.

    2014-01-01

    Axonal transport is essential for neuronal function, and many neurodevelopmental and neurodegenerative diseases result from mutations in the axonal transport machinery. Anterograde transport supplies distal axons with newly synthesized proteins and lipids, including synaptic components required to maintain presynaptic activity. Retrograde transport is required to maintain homeostasis by removing aging proteins and organelles from the distal axon for degradation and recycling of components. Retrograde axonal transport also plays a major role in neurotrophic and injury response signaling. This review provides an overview of the axonal transport pathway and discusses its role in neuronal function. PMID:25374356

  13. Modelling organelle transport after traumatic axonal injury.

    PubMed

    Kuznetsov, I A; Kuznetsov, A V

    2015-01-01

    This paper is motivated by recent experimental research (Tang-Schomer et al. 2012) on the formation of periodic varicosities in axons after traumatic brain injury (TBI). TBI leads to the formation of undulated distortions in the axons due to their dynamic deformation. These distortions result in the breakage of some microtubules (MTs) near the peaks of undulations. The breakage is followed by catastrophic MT depolymerisation around the broken ends. Although after relaxation axons regain their straight geometry, the structure of the axon after TBI is characterised by the presence of periodic regions where the density of MTs has been decreased due to depolymerisation. We modelled organelle transport in an axon segment with such a damaged MT structure and investigated how this structure affects the distributions of organelle concentrations and fluxes. The modelling results suggest that organelles accumulate at the boundaries of the region where the density of MTs has been decreased by depolymerisation. According to the model, the presence of such damaged regions decreases the organelle flux by only about 12%. This provides evidence that axon degradation after TBI may be caused by organelle accumulation rather than by starvation due to insufficient organelle flux.

  14. A model of axonal transport drug delivery

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Andrey V.

    2012-04-01

    In this paper a model of targeted drug delivery by means of active (motor-driven) axonal transport is developed. The model is motivated by recent experimental research by Filler et al. (A.G. Filler, G.T. Whiteside, M. Bacon, M. Frederickson, F.A. Howe, M.D. Rabinowitz, A.J. Sokoloff, T.W. Deacon, C. Abell, R. Munglani, J.R. Griffiths, B.A. Bell, A.M.L. Lever, Tri-partite complex for axonal transport drug delivery achieves pharmacological effect, Bmc Neuroscience 11 (2010) 8) that reported synthesis and pharmacological efficiency tests of a tri-partite complex designed for axonal transport drug delivery. The developed model accounts for two populations of pharmaceutical agent complexes (PACs): PACs that are transported retrogradely by dynein motors and PACs that are accumulated in the axon at the Nodes of Ranvier. The transitions between these two populations of PACs are described by first-order reactions. An analytical solution of the coupled system of transient equations describing conservations of these two populations of PACs is obtained by using Laplace transform. Numerical results for various combinations of parameter values are presented and their physical significance is discussed.

  15. Synaptic Democracy and Vesicular Transport in Axons

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Levien, Ethan

    2015-04-01

    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".

  16. Enhanced β-secretase processing alters APP axonal transport and leads to axonal defects

    PubMed Central

    Rodrigues, Elizabeth M.; Weissmiller, April M.; Goldstein, Lawrence S.B.

    2012-01-01

    Alzheimer's disease (AD) is a neurodegenerative disease pathologically characterized by amyloid plaques and neurofibrillary tangles in the brain. Before these hallmark features appear, signs of axonal transport defects develop, though the initiating events are not clear. Enhanced amyloidogenic processing of amyloid precursor protein (APP) plays an integral role in AD pathogenesis, and previous work suggests that both the Aβ region and the C-terminal fragments (CTFs) of APP can cause transport defects. However, it remains unknown if APP processing affects the axonal transport of APP itself, and whether increased APP processing is sufficient to promote axonal dystrophy. We tested the hypothesis that β-secretase cleavage site mutations of APP alter APP axonal transport directly. We found that the enhanced β-secretase cleavage reduces the anterograde axonal transport of APP, while inhibited β-cleavage stimulates APP anterograde axonal transport. Transport behavior of APP after treatment with β- or γ-secretase inhibitors suggests that the amount of β-secretase cleaved CTFs (βCTFs) of APP underlies these transport differences. Consistent with these findings, βCTFs have reduced anterograde axonal transport compared with full-length, wild-type APP. Finally, a gene-targeted mouse with familial AD (FAD) Swedish mutations to APP, which enhance the β-cleavage of APP, develops axonal dystrophy in the absence of mutant protein overexpression, amyloid plaque deposition and synaptic degradation. These results suggest that the enhanced β-secretase processing of APP can directly impair the anterograde axonal transport of APP and are sufficient to lead to axonal defects in vivo. PMID:22843498

  17. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy

    PubMed Central

    Lee, Sooyeon; Sato, Yutaka; Nixon, Ralph A.

    2012-01-01

    In the hallmark neuritic dystrophy of Alzheimer’s disease (AD), autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both axonal transport and autophagy. Here, we investigated the possibility that impaired lysosomal proteolysis could be a basis for both defects leading to neuritic dystrophy. In living primary mouse cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes forming in axons rapidly acquired the endo-lysosomal markers, Rab7 and LAMP1, and underwent exclusive retrograde movement. Proteolytic clearance of these transported autophagic vacuoles was initiated upon fusion with bi-directionally moving lysosomes that increase in number at more proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or by suppressing lysosomal acidification slowed the axonal transport of autolysosomes, late endosomes and lysosomes and caused their selective accumulation within dystrophic axonal swellings. Mitochondria and other organelles lacking cathepsins moved normally under these conditions, indicating that the general functioning of the axonal transport system was preserved. Dystrophic swellings induced by lysosomal proteolysis inhibition resembled in composition those in several mouse models of AD and also acquired other AD-like features, including immunopositivity for ubiquitin, APP, and neurofilament protein hyperphosphorylation. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn, largely cleared autophagic substrates and reversed the axonal dystrophy. These studies identify the AD-associated defects in neuronal lysosomal proteolysis as a possible basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with AD. PMID:21613495

  18. Kinesin-1/Hsc70-dependent mechanism of slow axonal transport and its relation to fast axonal transport

    PubMed Central

    Terada, Sumio; Kinjo, Masataka; Aihara, Makoto; Takei, Yosuke; Hirokawa, Nobutaka

    2010-01-01

    Cytoplasmic protein transport in axons (‘slow axonal transport') is essential for neuronal homeostasis, and involves Kinesin-1, the same motor for membranous organelle transport (‘fast axonal transport'). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin-1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ-like domain of the kinesin light chain in the Kinesin-1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin-1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant-negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction. PMID:20111006

  19. Fast axonal transport in isolated axoplasm from the squid giant axon.

    PubMed

    Song, Yuyu; Kang, Minsu; Morfini, Gerardo; Brady, Scott T

    2016-01-01

    The giant axon of the squid provides a unique cell biological model for analyzing the biochemistry and cell biology of the axon. These axons may exceed 500 μm in diameter and can be readily dissected. Once the surrounding small axons and connective tissue are removed, the axoplasm can be extruded as an intact cylinder of isolated cytoplasm. This isolated axoplasm is morphologically indistinguishable from the intact axon, but without permeability barriers. Fast axonal transport will continue for more than 4 h after extrusion and can be visualized in real time. By perfusing defined concentrations of proteins and/or reagents into the axoplasm, this preparation represents a powerful model for study of intracellular trafficking and its underlying molecular mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury.

    PubMed

    Tang-Schomer, Min D; Johnson, Victoria E; Baas, Peter W; Stewart, William; Smith, Douglas H

    2012-01-01

    Due to their viscoelastic nature, white matter axons are susceptible to damage by high strain rates produced during traumatic brain injury (TBI). Indeed, diffuse axonal injury (DAI) is one of the most common features of TBI, characterized by the hallmark pathological profiles of axonal bulbs at disconnected terminal ends of axons and periodic swellings along axons, known as "varicosities." Although transport interruption underlies axonal bulb formation, it is unclear how varicosities arise, with multiple sites accumulating transported materials along one axon. Recently, axonal microtubules have been found to physically break during dynamic stretch injury of cortical axons in vitro. Here, the same in vitro model was used in parallel with histopathological analyses of human brains acquired acutely following TBI to examine the potential role of mechanical microtubule damage in varicosity formation post-trauma. Transmission electron microscopy (TEM) following in vitro stretch injury revealed periodic breaks of individual microtubules along axons that regionally corresponded with undulations in axon morphology. However, typically less than a third of microtubules were broken in any region of an axon. Within hours, these sites of microtubule breaks evolved into periodic swellings. This suggests axonal transport may be halted along one broken microtubule, yet can proceed through the same region via other intact microtubules. Similar axonal undulations and varicosities were observed following TBI in humans, suggesting primary microtubule failure may also be a feature of DAI. These data indicate a novel mechanism of mechanical microtubule damage leading to partial transport interruption and varicosity formation in traumatic axonal injury.

  1. The transport properties of axonal microtubules establish their polarity orientation

    PubMed Central

    1993-01-01

    It is well established that axonal microtubules (MTs) are uniformly oriented with their plus ends distal to the neuronal cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981. J. Cell Biol. 91:661-665). However, the mechanisms by which these MTs achieve their uniform polarity orientation are unknown. Current models for axon growth differ with regard to the contributions of MT assembly and transport to the organization and elaboration of the axonal MT array. Do the transport properties or assembly properties of axonal MTs determine their polarity orientation? To distinguish between these possibilities, we wished to study the initiation and outgrowth of axons under conditions that would arrest MT assembly while maintaining substantial levels of preexisting polymer in the cell body that could still be transported into the axon. We found that we could accomplish this by culturing rat sympathetic neurons in the presence of nanomolar levels of vinblastine. In concentrations of the drug up to and including 100 nM, the neurons actively extend axons. The vinblastine- axons are shorter than control axons, but clearly contain MTs. To quantify the effects of the drug on MT mass, we compared the levels of polymer throughout the cell bodies and axons of neurons cultured overnight in the presence of 0, 16, and 50 nM vinblastine with the levels of MT polymer in freshly plated neurons before axon outgrowth. Without drug, the total levels of polymer increase by roughly twofold. At 16 nM vinblastine, the levels of polymer are roughly equal to the levels in freshly plated neurons, while at 50 nM, the levels of polymer are reduced by about half this amount. Thus, 16 nM vinblastine acts as a "kinetic stabilizer" of MTs, while 50 nM results in some net MT disassembly. At both drug concentrations, there is a progressive increase in the levels of MT polymer in the axons as they grow, and a corresponding depletion of polymer from the cell body. These results indicate that

  2. Cytoplasmic dynein is associated with slow axonal transport.

    PubMed Central

    Dillman, J F; Dabney, L P; Pfister, K K

    1996-01-01

    Neuronal function is dependent on the transport of materials from the cell body to the synapse via anterograde axonal transport. Anterograde axonal transport consists of several components that differ in both rate and protein composition. In fast transport, membranous organelles are moved along microtubules by the motor protein kinesin. The cytoskeleton and the cytomatrix proteins move in the two components of slow transport. While the mechanisms underlying slow transport are unknown, it has been hypothesized that the movement of microtubules in slow transport is generated by sliding. To determine whether dynein, a motor protein that causes microtubule sliding in flagella, may play a role in slow axonal transport, we identified the transport rate components with which cytoplasmic dynein is associated in rat optic nerve. Nearly 80% of the anterogradely moving dynein was associated with slow transport, whereas only approximately 15% of the dynein was associated with the membranous organelles of anterograde fast axonal transport. A segmental analysis of the transport of dynein through contiguous regions of the optic nerve and tract showed that dynein is associated with the microfilaments and other proteins of slow component b. Dynein from this transport component has the capacity to bind microtubules in vitro. These results are consistent with the hypothesis that cytoplasmic dynein generates the movement of microtubules in slow axonal transport. A model is presented to illustrate how dynein attached to the slow component b complex of proteins is appropriately positioned to generate force of the correct polarity to slide microtubules down the axon. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8552592

  3. Axonal Transport and Morphology: How Myelination gets Nerves into Shape

    NASA Astrophysics Data System (ADS)

    Jung, Peter; Zhao, Peng; Monsma, Paula; Brown, Tony

    2011-03-01

    The local caliber of mature axons is largely determined by neurofilament (NF) content. The axoskeleton, mainly consisting of NFs, however, is dynamic. NFs are assembled in the cell body and are transported by molecular motors on microtubule tracks along the axon at a slow rate of fractions of mm per day. We combine live cell fluorescent imaging techniques to access NF transport in myelinated and non-myelinated segments of axons with computational modeling of the active NF flow to show that a), myelination locally slows NF transport rates by regulating duty ratios and b), that the predicted increase in axon caliber agrees well with experiments. This study, for the first time, links NF kinetics directly to axonal morphology, providing a novel conceptual framework for the physical understanding of processes leading to the formation of axonal structures such as the ``Nodes of Ranvier'' as well as abnormal axonal swellings associated with neurodegenerative diseases like Amyotrophic lateral sclerosis (ALS). NSF grants # IOS-0818412(PJ) and IOS-0818653 (AB).

  4. Axonal transport deficits in multiple sclerosis: spiraling into the abyss.

    PubMed

    van den Berg, Robert; Hoogenraad, Casper C; Hintzen, Rogier Q

    2017-07-01

    The transport of mitochondria and other cellular components along the axonal microtubule cytoskeleton plays an essential role in neuronal survival. Defects in this system have been linked to a large number of neurological disorders. In multiple sclerosis (MS) and associated models such as experimental autoimmune encephalomyelitis (EAE), alterations in axonal transport have been shown to exist before neurodegeneration occurs. Genome-wide association (GWA) studies have linked several motor proteins to MS susceptibility, while neuropathological studies have shown accumulations of proteins and organelles suggestive for transport deficits. A reduced effectiveness of axonal transport can lead to neurodegeneration through inhibition of mitochondrial motility, disruption of axoglial interaction or prevention of remyelination. In MS, demyelination leads to dysregulation of axonal transport, aggravated by the effects of TNF-alpha, nitric oxide and glutamate on the cytoskeleton. The combined effect of all these pathways is a vicious cycle in which a defective axonal transport system leads to an increase in ATP consumption through loss of membrane organization and a reduction in available ATP through inhibition of mitochondrial transport, resulting in even further inhibition of transport. The persistent activity of this positive feedback loop contributes to neurodegeneration in MS.

  5. Action in the axon: generation and transport of signaling endosomes.

    PubMed

    Cosker, Katharina E; Courchesne, Stephanie L; Segal, Rosalind A

    2008-06-01

    Neurons extend axonal processes over long distances, necessitating efficient transport mechanisms to convey target-derived neurotrophic survival signals from remote distal axons to cell bodies. Retrograde transport, powered by dynein motors, supplies cell bodies with survival signals in the form of 'signaling endosomes'. In this review, we will discuss new advances in our understanding of the motor proteins that bind to and move signaling components in a retrograde direction and discuss mechanisms that might specify distinct neuronal responses to spatially restricted neurotrophin signals. Disruption of retrograde transport leads to a variety of neurodegenerative diseases, highlighting the role of retrograde transport of signaling endosomes for axonal maintenance and the importance of efficient transport for neuronal survival and function.

  6. Axonal degeneration in paraplegin-deficient mice is associated with abnormal mitochondria and impairment of axonal transport

    PubMed Central

    Ferreirinha, Fatima; Quattrini, Angelo; Pirozzi, Marinella; Valsecchi, Valentina; Dina, Giorgia; Broccoli, Vania; Auricchio, Alberto; Piemonte, Fiorella; Tozzi, Giulia; Gaeta, Laura; Casari, Giorgio; Ballabio, Andrea; Rugarli, Elena I.

    2004-01-01

    In several neurodegenerative diseases, axonal degeneration occurs before neuronal death and contributes significantly to patients’ disability. Hereditary spastic paraplegia (HSP) is a genetically heterogeneous condition characterized by selective degeneration of axons of the corticospinal tracts and fasciculus gracilis. HSP may therefore be considered an exemplary disease to study the local programs mediating axonal degeneration. We have developed a mouse model for autosomal recessive HSP due to mutations in the SPG7 gene encoding the mitochondrial ATPase paraplegin. Paraplegin-deficient mice are affected by a distal axonopathy of spinal and peripheral axons, characterized by axonal swelling and degeneration. We found that mitochondrial morphological abnormalities occurred in synaptic terminals and in distal regions of axons long before the first signs of swelling and degeneration and correlated with onset of motor impairment during a rotarod test. Axonal swellings occur through massive accumulation of organelles and neurofilaments, suggesting impairment of anterograde axonal transport. Retrograde axonal transport is delayed in symptomatic mice. We speculate that local failure of mitochondrial function may affect axonal transport and cause axonal degeneration. Our data suggest that a timely therapeutic intervention may prevent the loss of axons. PMID:14722615

  7. Axonal transport of thiamine in frog sciatic nerves in vitro.

    PubMed

    Bergquist, J E; Hanson, M

    1983-03-01

    Thiamine has an essential and unknown function in nerve membranes. Administration of thiamine can alleviate symptoms of thiamine deficiency within a few hours. The time course is consistent with a fast axonal transport of the vitamin. Very little is known about axonal transport of low-molecular-weight substances with a preferential localization to the axon membrane. We investigated if labeled thiamine could be transported in the frog sciatic nerve. Radioactivity accumulated proximal to a ligature on the sciatic nerve after supplying the dorsal ganglia with [35S]thiamine in vitro. The accumulation was reduced by inhibition of the energy metabolism with dinitrophenol and by inhibition of protein synthesis in the ganglia with cycloheximide. Vinblastine did not affect the accumulation of thiamine at a concentration which was sufficient to block transport of [3H]leucine-labeled proteins. Accumulation distal to a ligature could be demonstrated in vivo but not in vitro after injecting the gastrocnemius muscle with labeled thiamine. Axonal transport of [3H]leucine-labeled proteins was inhibited by thiamine at millimolar concentrations in the incubation medium. A transient reduction of the compound action potential was obtained at these concentrations. Thiamine was migrating at a fast rate in frog sciatic nerves in both orthograde and retrograde directions. The uptake and/or transport was dependent on energy metabolism and a concomitant protein synthesis. The lack of effect by vinblastine suggests that the transported fraction of thiamine differs in subcellular localization from the bulk of transported [3H]leucine-labeled proteins.

  8. Partial Interruption of Axonal Transport Due to Microtubule Breakage Accounts for the Formation of Periodic Varicosities after Traumatic Axonal Injury

    PubMed Central

    Tang-Schomer, Min D.; Johnson, Victoria E.; Baas, Peter W.; Stewart, William; Smith, Douglas H.

    2012-01-01

    Due to their viscoelastic nature, white matter axons are susceptible to damage by high strain rates produced during traumatic brain injury (TBI). Indeed, diffuse axonal injury (DAI) is one of the most common features of TBI, characterized by the hallmark pathological profiles of axonal bulbs at disconnected terminal ends of axons and periodic swellings along axons, known as “varicosities.” Although transport interruption underlies axonal bulb formation, it is unclear how varicosities arise, with multiple sites accumulating transported materials along one axon. Recently, axonal microtubules have been found to physically break during dynamic stretch-injury of cortical axons in vitro. Here, the same in vitro model was used in parallel with histopathological analyses of human brains acquired acutely following TBI to examine the potential role of mechanical microtubule damage in varicosity formation post-trauma. Transmission electron microscopy (TEM) following in vitro stretch-injury revealed periodic breaks of individual microtubules along axons that regionally corresponded with undulations in axon morphology. However, typically less than a third of microtubules were broken in any region of an axon. Within hours, these sites of microtubule breaks evolved into periodic swellings. This suggests axonal transport may be halted along one broken microtubule, yet can proceed through the same region via other intact microtubules. Similar axonal undulations and varicosities were observed following TBI in humans, suggesting primary microtubule failure may also be a feature of DAI. These data indicate a novel mechanism of mechanical microtubule damage leading to partial transport interruption and varicosity formation in traumatic axonal injury. PMID:22079153

  9. Retrograde axonal transport of VZV: kinetic studies in hESC-derived neurons.

    PubMed

    Grigoryan, Sergei; Kinchington, Paul R; Yang, In Hong; Selariu, Anca; Zhu, Hua; Yee, Michael; Goldstein, Ronald S

    2012-12-01

    Retrograde axonal transport of the neurotropic alphaherpesvirus Varicella zoster virus (VZV) from vesicles at the skin results in sensory neuron infection and establishment of latency. Reactivation from latency leads to painful herpes zoster. The lack of a suitable animal model of these processes for the highly human-restricted VZV has resulted in a dearth of knowledge regarding the axonal transport of VZV. We recently demonstrated VZV infection of distal axons, leading to subsequent capsid transport to the neuronal somata, and replication and release of infectious virus using a new model based on neurons derived from human embryonic stem cells (hESC). In the present study, we perform a kinetic analysis of the retrograde transport of green fluorescent protein-tagged ORF23 in VZV capsids using hESC-derived neurons compartmentalized microfluidic chambers and time-lapse video microscopy. The motion of the VZV was discontinuous, showing net retrograde movement with numerous short pauses and reversals in direction. Velocities measured were higher 1 h after infection than 6 h after infection, while run lengths were similar at both time points. The hESC-derived neuron model was also used to show that reduced neuronal spread by a VZV loss-of-function mutant for ORF7 is not due to the prevention of axonal infection and transport of the virus to the neuronal somata. hESC-derived neurons are, therefore, a powerful model for studying axonal transport of VZV and molecular characteristics of neuronal infection.

  10. Mechanistic logic underlying the axonal transport of cytosolic proteins

    PubMed Central

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

    2011-01-01

    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

  11. In vivo imaging of axonal transport using MRI: aging and Alzheimer's disease.

    PubMed

    Minoshima, Satoshi; Cross, Donna

    2008-03-01

    MRI using manganese as a trans-synaptic axonal tracing agent can unveil dynamics of axonal transport in living subjects. We use this technology to test the hypotheses if impaired axonal transport is a significant pathophysiological process in aging and early Alzheimer's disease (AD) and in part accounting for "selective vulnerability" of projection neurons in AD. To allow quantitative assessment of axonal transport in vivo, we developed voxel-based statistical mapping technology as well as a tracer kinetic modeling method based on mass transport for manganese-enhanced MRI to estimate axonal transport rates in aging rats and AD transgenic mice. These techniques demonstrated manganese-enhanced signal changes in axonal projections of the olfactory tract and decreased axonal transport rates in rodent models of aging and AD. Altered axonal transport may be a critical pathophysiological process in aging and AD. Manganese-enhanced MRI provides exciting opportunities for the investigations of altered axonal transport in AD and related disorders.

  12. Fast axonal transport by neurons from the jellyfish Cyanea capillata.

    PubMed

    Anderson, P A; Schwab, W E; Gilbert, S; Allen, R D

    1986-01-01

    Neurons of the motor nerve net of Cyanea capillata were examined using video-enhanced DIC optics. A variety of organelles were visible within the axons and many were mobile. To quantify the movement organelles were divided into three classes (large, medium, and small) and the rates, direction, and types of movement displayed by the different particle types examined. The overall behavior and rates of movement of transported particles were comparable with those in axons from other species. The largest particles, mainly mitochondria were the slowest moving but were the only particles to reverse their direction of movement or to undergo interactions with other particles. The fastest movement was by the small particles, but both they and medium sized particles were transported continuously. In addition, the linear elements in these axons underwent considerable lateral movement.

  13. Rotational dynamics of cargos at pauses during axonal transport

    SciTech Connect

    Gu, Yan; Sun, Wei; Wang, Gufeng; Jeftinija, Ksenija; Jeftinija, Srdija; Fang, Ning

    2012-08-28

    Direct visualization of axonal transport in live neurons is essential for our understanding of the neuronal functions and the working mechanisms of microtubule-based motor proteins. Here we use the high-speed single particle orientation and rotational tracking technique to directly visualize the rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport, with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause.

  14. Characterization of axonal transport defects in Drosophila Huntingtin mutants.

    PubMed

    Weiss, Kurt R; Littleton, J Troy

    Polyglutamine (polyQ) expansion within Huntingtin (Htt) causes the fatal neurodegenerative disorder Huntington's Disease (HD). Although Htt is ubiquitously expressed and conserved from Drosophila to humans, its normal biological function is still being elucidated. Here we characterize a role for the Drosophila Htt homolog (dHtt) in fast axonal transport (FAT). Generation and expression of transgenic dHtt-mRFP and human Htt-mRFP fusion proteins in Drosophila revealed co-localization with mitochondria and synaptic vesicles undergoing FAT. However, Htt was not ubiquitously associated with the transport machinery, as it was excluded from dense-core vesicles and APLIP1 containing vesicles. Quantification of cargo movement in dHtt deficient axons revealed that mitochondria and synaptic vesicles show a decrease in the distance and duration of transport, and an increase in the number of pauses. In addition, the ratio of retrograde to anterograde flux was increased in mutant animals. Dense-core vesicles did not display similar defects in processivity, but did show altered retrograde to anterograde flux along axons. Given the co-localization with mitochondria and synaptic vesicles, but not dense-core vesicles, the data suggest dHtt likely acts locally at cargo interaction sites to regulate processivity. An increase in dynein heavy chain expression was also observed in dHtt mutants, suggesting that the altered flux observed for all cargo may represent secondary transport changes occurring independent of dHtt's primary function. Expression of dHtt in a milton (HAP1) mutant background revealed that the protein does not require mitochondria or HAP1 to localize along axons, suggesting Htt has an independent mechanism for coupling with motors to regulate their processivity during axonal transport.

  15. Depression of fast axonal transport in axons demyelinated by intraneural injection of a neurotoxin from K. humboldtiana.

    PubMed

    Muñoz-Martínez, E J; Cuéllar-Pedroza, L H; Rubio-Franchini, C; Jáuregui-Rincón, J; Joseph-Nathan, P

    1994-11-01

    Tullidinol, a neurotoxin extracted from the Karwinskia humboldtiana fruit, dissolved in peanut oil was injected into the right sciatic nerve of adult cats. The contralateral sciatic nerve received an equivalent volume of peanut oil alone. The fast axonal transport of labeled ([3H]Leucine) protein was studied in sensory and motor axons of both sciatic nerves. The radioactive label was pressure injected either into the L7 dorsal root ganglion or the ventral region of the same spinal cord segment. Several days after the toxin injection, the cat limped and the Achilles tendon reflex was nearly absent in the right hind limb. The amount of transported label was decreased distal to the site of toxin injection. Proximal to this site, the transported material was damned. Sensory and motor axons showed similar changes. In addition, the toxin produced demyelination and axonal degeneration. Axonal transport and the structure of the axons were normal in the contralateral nerve. Both, Schwann cells and axons of the right sciatic nerve showed globular inclusions, presumably oil droplets containing the toxin. We conclude that Schwann cells and axons as well are tullidinol targets.

  16. Slowing of the axonal transport of neurofilament proteins during development

    SciTech Connect

    Hoffman, P.N.; Lasek, R.J.; Griffin, J.W.; Price, D.L.

    1983-08-01

    We examined age-dependent changes in neurofilament transport in motor axons of the rat sciatic nerve. SDS-PAGE and gel fluorography confirmed that the distribution of labeled neurofilament triplet protein coincides with the major slow component a (SCa) wave in these neurons. The velocity of neurofilament transport was calculated on the basis of the location of the 50th percentile of radioactivity in this wave 33 days after motor neurons were labeled by the intraspinal administration of (/sup 3/H)leucine and (/sup 3/H)lysine. Overall, the velocity fell from 1.95 mm/day at 3 weeks of age to 1.12 mm/day at 20 weeks. Between 3 and 10 weeks, it fell at a 6-fold higher rate (0.096 mm/day/week) than between 10 and 20 weeks (0.016 mm/day/week). We also found a marked change in the shape of the slow component wave during development. It appeared to consist of several overlapping peaks moving at slightly different velocities in animals 10 weeks of age or less as compared to a single slower moving peak at 20 weeks. We propose that the velocity of slow axonal transport reflects the level of maturation of the neuron, and that the presence of several overlapping peaks of transported radioactivity in the sciatic nerve of younger animals reflects the presence of several populations of motor axons at different stages of development. We also discuss the relationship between changes in the velocity of neurofilament transport and alterations in the composition of the cytoskeleton that occur as the axon grows in caliber during postnatal development.

  17. Monte-Carlo Study of Axonal Transport in a Neuron

    NASA Astrophysics Data System (ADS)

    Shrestha, Uttam; Yu, Clare; Jia, Zhiyuan; Erickson, Robert; Gross, Steven

    2011-03-01

    A living cell has an infrastructure much like that of a city. A key component is the transportation system that consists of roads (filaments) and molecular motors (proteins) that haul cargo along these roads. We will present a Monte Carlo simulation of intracellular transport inside an axon in which motor proteins carry cargos along microtubules and are able to switch from one microtubule to another. The breakdown of intracellular transport in neurons has been associated with neurodegenerative diseases such as Alzheimer's, Lou Gehig's disease (ALS), and Huntingdon's disease. This work was supported by NIGMS grant number 5R01GM79156.

  18. Axonal Transport Rates In Vivo Are Unaffected by Tau Deletion or Overexpression in Mice

    PubMed Central

    Yuan, Aidong; Kumar, Asok; Peterhoff, Corrinne; Duff, Karen; Nixon, Ralph A.

    2010-01-01

    Elevated tau expression has been proposed as a possible basis for impaired axonal transport in Alzheimer’s disease. To address this hypothesis, we analyzed the movement of pulse radiolabeled proteins in vivo along retinal ganglion cell (RGC) axons of mice that lack tau or overexpress human tau isoforms. Here, we show that the global axonal transport rates of slow and fast transport cargoes in axons are not significantly impaired when tau expression is eliminated or increased. In addition, markers of slow transport (neurofilament light subunit) and fast transport (snap25) do not accumulate in retinas and are distributed normally along optic axons in mice that lack or overexpress tau. Finally, ultrastructural analyses revealed no abnormal accumulations of vesicular organelles or neurofilaments in RGC perikarya or axons in mice overexpressing or lacking tau. These results suggest that tau is not essential for axonal transport and that transport rates in vivo are not significantly affected by substantial fluctuations in tau expression. PMID:18272688

  19. Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells

    PubMed Central

    1981-01-01

    In an effort to understand the regulation of the transition of a mature neuron to the growth, or regenerating, state we have analyzed the composition of the axonally transported proteins in the retinal ganglion cells of the toad Bufo marinus after inducing axon regeneration by crushing the optic nerve. At increasing intervals after axotomy, we labeled the retinal ganglion cells with [35S]methionine and subsequently analyzed the labeled transported polypeptides in the crushed optic nerve by means of one- and two-dimensional electrophoretic techniques. The most significant conclusion from these experiments is that, while the transition from the mature to the regenerating state does not require a gross qualitative alteration in the composition of axonally transported proteins, the relative labeling of a small subset of rapidly transported proteins is altered dramatically (changes of more than 20-fold) and reproducibly (more than 30 animals) by axotomy. One of these growth-associated proteins (GAPs) was soluble in an aqueous buffer, while three were associated with a crude membrane fraction. The labeling of all three of the membrane- associated GAPs increased during the first 8 d after axotomy, and they continued to be labeled for at least 4 wk. The modulation of these proteins after axotomy is consistent with the possibility that they are involve in growth-specific functions and that the altered expression of a small number of genes is a crucial regulatory event in the transition of a mature neuron to a growth state. In addition to these selective changes in rapidly transported proteins, we observed the following more general metabolic correlates of the regeneration process: The total radioactive label associated with the most rapidly transported proteins (groups I and II) increased three to fourfold during the first 8 d after the nerve was crushed, while the total label associated with more slowly moving proteins (group IV) increased about 10-fold during this same

  20. Axonal guidance by surface microstructuring for intracellular transport investigations.

    PubMed

    Pelzl, Carina; Arcizet, Delphine; Piontek, Guido; Schlegel, Jürgen; Heinrich, Doris

    2009-11-09

    Intracellular transport, a complex interplay of diverse processes, is fundamental for the development, function and survival of cells. Passive diffusion and active transport phases alternate in living cells, with active phases arising from molecular motors, such as kinesin or dynein, pulling cargoes along microtubules. A better understanding of stochasic mechanisms involved in motor-microtubule interactions and in diffusion processes, which enable efficient active transport over long distances in motor neurons, requires a better link between theoretical models and live-cell experiments. Herein, we establish one-dimensional (1D) intracellular transport geometries, suitable for comparing experimental findings with recent theoretical 1D model predictions, by guiding axonal outgrowth of pheochromocytoma (PC12) cells along predefined chemical surface structures with a strip width of 2 microm, fabricated by means of microscale plasma-initiated patterning (microPIP method). Quantification of the intracellular transport of quantum dots (QDs) in straight axons, which exhibit almost parallel microtubules, is obtained by our recently developed algorithm based on a time-resolved mean-square displacement (MSD) analysis. Such a thorough dissection of experimental data will be useful for validation and clarification of current theoretical transport models.

  1. Kinesin light chains are essential for axonal transport in Drosophila.

    PubMed

    Gindhart, J G; Desai, C J; Beushausen, S; Zinn, K; Goldstein, L S

    1998-04-20

    Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075-1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (. Cell 64:1093-1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075-1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.

  2. Axonal transport in the electromotor nerves of Torpedo marmorata.

    PubMed

    Davies, L P; Whittaker, V P; Zimmermann, H

    1977-12-19

    Studies on the axonal transport of cholinergic cell components were made on the electromotor nerves of Torpedo marmorata. Choline acetyltransferase was rapidly accumulated at ligatures on Torpedo nerves, both in vivo and in segments incubated in vitro. In vivo accumulation was maximal approximately one month after nerve interruption. Orthograde transport (both in vitro and in vivo) is calculated to have a velocity of 50--140 mm/day, if, as double-ligature experiments suggest, only about 15% of the axoplasmic enzyme is mobile. A small retrograde accumulation of the transferase was demonstrated. Lactate dehydrogenase did not accumulate but a slight reduction of its activity at ligatures was observed. In contrast to mammalian cholinergic nerves, no accumulation of esterase was observed. ACh accumulation proximal to a cut was apparent and may result in part from local synthesis in the presence of elevated levels of its synthesizing enzyme. Measurements have been made on the activity of choline acetyltransferase in the brain and all parts of the "electric system". In view of these results it is difficult to see how the measured rate of axonal translocation is sufficient to supply the levels of the enzyme found within the electric organ. Within the electromotor cells, choline acetyltransferase is highly concentrated in the axon terminals.

  3. Rapid axonal transport in primate optic nerve. Distribution of pressure-induced interruption.

    PubMed

    Radius, R L; Anderson, D R

    1981-04-01

    Six primate eyes were studied after four hours of elevated intraocular pressure. Tissue specimens from the region of the lamina cribrosa were examined in cross section by transmission electron microscopy. Interruption in fast orthograde and retrograde axonal transport was identified in individual axons by noting accumulation of membraneous microorganelles, such as mitochondria and microvesicles within axon cylinders. Although organelle accumulation varied from bundle to bundle, involvement of individual axons was diffuse across the extent of a specific axon bundle. This observation contradicts the apparent association of axonal transport block with crosswise-oriented trabecular beams at the level of the lamina cribrosa as seen in tissue specimens examined in longitudinal section. It also fails to support the notion that blocked axonal transport with elevated pressure is produced by kinking of axons at the lamina.

  4. Axonal Transport: How High Microtubule Density Can Compensate for Boundary Effects in Small-Caliber Axons

    PubMed Central

    Wortman, Juliana C.; Shrestha, Uttam M.; Barry, Devin M.; Garcia, Michael L.; Gross, Steven P.; Yu, Clare C.

    2014-01-01

    Long-distance intracellular axonal transport is predominantly microtubule-based, and its impairment is linked to neurodegeneration. In this study, we present theoretical arguments that suggest that near the axon boundaries (walls), the effective viscosity can become large enough to impede cargo transport in small (but not large) caliber axons. Our theoretical analysis suggests that this opposition to motion increases rapidly as the cargo approaches the wall. We find that having parallel microtubules close enough together to enable a cargo to simultaneously engage motors on more than one microtubule dramatically enhances motor activity, and thus minimizes the effects of any opposition to transport. Even if microtubules are randomly placed in axons, we find that the higher density of microtubules found in small-caliber axons increases the probability of having parallel microtubules close enough that they can be used simultaneously by motors on a cargo. The boundary effect is not a factor in transport in large-caliber axons where the microtubule density is lower. PMID:24559984

  5. Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides

    PubMed Central

    Barkus, Rosemarie V.; Klyachko, Olga; Horiuchi, Dai; Dickson, Barry J.

    2008-01-01

    A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism. PMID:17989365

  6. The dynein inhibitor Ciliobrevin D inhibits the bidirectional transport of organelles along sensory axons and impairs NGF-mediated regulation of growth cones and axon branches.

    PubMed

    Sainath, Rajiv; Gallo, Gianluca

    2015-07-01

    The axonal transport of organelles is critical for the development, maintenance, and survival of neurons, and its dysfunction has been implicated in several neurodegenerative diseases. Retrograde axon transport is mediated by the motor protein dynein. In this study, using embryonic chicken dorsal root ganglion neurons, we investigate the effects of Ciliobrevin D, a pharmacological dynein inhibitor, on the transport of axonal organelles, axon extension, nerve growth factor (NGF)-induced branching and growth cone expansion, and axon thinning in response to actin filament depolymerization. Live imaging of mitochondria, lysosomes, and Golgi-derived vesicles in axons revealed that both the retrograde and anterograde transport of these organelles was inhibited by treatment with Ciliobrevin D. Treatment with Ciliobrevin D reversibly inhibits axon extension and transport, with effects detectable within the first 20 min of treatment. NGF induces growth cone expansion, axonal filopodia formation and branching. Ciliobrevin D prevented NGF-induced formation of axonal filopodia and branching but not growth cone expansion. Finally, we report that the retrograde reorganization of the axonal cytoplasm which occurs on actin filament depolymerization is inhibited by treatment with Ciliobrevin D, indicating a role for microtubule based transport in this process, as well as Ciliobrevin D accelerating Wallerian degeneration. This study identifies Ciliobrevin D as an inhibitor of the bidirectional transport of multiple axonal organelles, indicating this drug may be a valuable tool for both the study of dynein function and a first pass analysis of the role of axonal transport.

  7. Effect of kinesin velocity distribution on slow axonal transport

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Andrey

    2012-08-01

    The goal of this paper is to investigate the effect that a distribution of kinesin motor velocities could have on cytoskeletal element (CE) concentration waves in slow axonal transport. Previous models of slow axonal transport based on the stop-and-go hypothesis (P. Jung, A. Brown, Modeling the slowing of neurofilament transport along the mouse sciatic nerve, Physical Biology 6 (2009) 046002) assumed that in the anterograde running state all CEs move with one and the same velocity as they are propelled by kinesin motors. This paper extends the aforementioned theoretical approach by allowing for a distribution of kinesin motor velocities; the distribution is described by a probability density function (PDF). For a two kinetic state model (that accounts for the pausing and running populations of CEs) an analytical solution describing the propagation of the CE concentration wave is derived. Published experimental data are used to obtain an analytical expression for the PDF characterizing the kinesin velocity distribution; this analytical expression is then utilized as an input for computations. It is demonstrated that accounting for the kinesin velocity distribution increases the rate of spreading of the CE concentration waves, which is a significant improvement in the two kinetic state model.

  8. Neurogenetics of slow axonal transport: from cells to animals.

    PubMed

    Sadananda, Aparna; Ray, Krishanu

    2012-09-01

    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.

  9. Axonal transport interruption and anatomy at the lamina cribrosa.

    PubMed

    Radius, R L; Bade, B

    1982-10-01

    Pressure-induced, focal axonal transport abnormalities were studied in 14 cat eyes by the examination of serial step-section tissue radioautogram. Although the patterns of the transport interruption at the lamina cribrosa varied from eye to eye, the temporal sectors of the nerve head were most often involved by this abnormality. The anatomy at the lamina cribrosa was studied in adjacent (6 micrometers) cross-sectional specimens. The thickness of the extra-bundle trabeculae and the nerve fiber bundle dimensions including the cross-sectional area and the number and the shape (the ratio of the major and the minor axis diameters) of the laminar pores were measured by computer-assisted perimeter analysis. There was no correlation between the location of the transport interruption and any of these anatomic measurements.

  10. Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons.

    PubMed

    Terada, S; Kinjo, M; Hirokawa, N

    2000-09-29

    Slow axonal transport depends on an active mechanism that conveys cytosolic proteins. To investigate its molecular mechanism, we now constructed an in vitro experimental system for observation of tubulin transport, using squid giant axons. After injecting fluorescence-labeled tubulin into the axons, we monitored the movement of fluorescence by confocal laser scanning microscopy and fluorescence correlation spectroscopy. Here, from the pharmacological experiments and the functional blocking of kinesin motor protein by anti-kinesin antibody, we show that the directional movement of fluorescent profile was dependent on kinesin motor function. The fluorescent correlation function and estimated translational diffusion time revealed that tubulin molecule was transported in a unique form of large transporting complex distinct from those of stable polymers or other cytosolic protein.

  11. Pathogenic Forms of Tau Inhibit Kinesin-Dependent Axonal Transport through a Mechanism Involving Activation of Axonal Phosphotransferases

    PubMed Central

    Kanaan, Nicholas M.; Morfini, Gerardo A.; LaPointe, Nichole E.; Pigino, Gustavo F.; Patterson, Kristina R.; Song, Yuyu; Andreadis, Athena; Fu, Yifan; Brady, Scott T.; Binder, Lester I.

    2012-01-01

    Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer’s disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2–18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau. PMID:21734277

  12. Pathogenic forms of tau inhibit kinesin-dependent axonal transport through a mechanism involving activation of axonal phosphotransferases.

    PubMed

    Kanaan, Nicholas M; Morfini, Gerardo A; LaPointe, Nichole E; Pigino, Gustavo F; Patterson, Kristina R; Song, Yuyu; Andreadis, Athena; Fu, Yifan; Brady, Scott T; Binder, Lester I

    2011-07-06

    Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2-18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau.

  13. Endothelin-1 impairs retrograde axonal transport and leads to axonal injury in rat optic nerve.

    PubMed

    Taniguchi, Takazumi; Shimazawa, Masamitsu; Sasaoka, Masaaki; Shimazaki, Atsushi; Hara, Hideaki

    2006-05-01

    The purpose of this study was to examine the effects of endothelin-1 (ET-1) on retrograde axonal transport in the rat optic nerve. Vehicle or ET-1 (0.2, 1, or 5 pmol/eye) were injected into the vitreous body in Sprague-Dawley rats. Retinal vessels were observed, using a fundus camera, before, and at 10 min, 3 days and 7 days after a single intravitreous injection. Two days after the injection, a neuronal tracer, fluoro gold, was administered via the superior colliculi to retrogradely label active retinal ganglion cells (RGCs). Five days after the tracer administration, retrogradely labeled RGCs were evaluated in the flat-mounted retina, and cross sections from each optic nerve were graded for injury by four independent, masked observers. ET-1 at 5 pmol/eye caused a significant constriction of retinal vessels (versus the vehicle-treated group) at 10 min after the injection. Intravitreous injection of ET-1 caused a dose-related decrease in the number of retrogradely labeled RGCs. Injection of 5 pmol/eye ET-1 led to a statistically significant decrease in the number of retrogradely labeled RGCs (versus the vehicle-treated group). ET-1 at 1 and 5 pmol/eye caused histological optic nerve damage (evaluated using a graded scale). The histological optic nerve damage correlated with the number of retrogradely labeled RGCs. In conclusion, a single intravitreous injection of ET-1 impaired retrograde axonal transport in the rat optic nerve and this impairment correlated with the histological optic nerve damage.

  14. Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase

    PubMed Central

    Morfini, Gerardo A.; Bosco, Daryl A.; Brown, Hannah; Gatto, Rodolfo; Kaminska, Agnieszka; Song, Yuyu; Molla, Linda; Baker, Lisa; Marangoni, M. Natalia; Berth, Sarah; Tavassoli, Ehsan; Bagnato, Carolina; Tiwari, Ashutosh; Hayward, Lawrence J.; Pigino, Gustavo F.; Watterson, D. Martin; Huang, Chun-Fang; Banker, Gary; Brown, Robert H.; Brady, Scott T.

    2013-01-01

    Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS. PMID:23776455

  15. Investigating the Slow Axonal Transport of Neurofilaments: A Precursor for Optimal Neuronal Signaling

    NASA Astrophysics Data System (ADS)

    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

  16. Tau phosphorylation affects its axonal transport and degradation

    PubMed Central

    Rodríguez-Martín, Teresa; Cuchillo-Ibáñez, Inmaculada; Noble, Wendy; Nyenya, Fanon; Anderton, Brian H.; Hanger, Diane P.

    2013-01-01

    Phosphorylated forms of microtubule-associated protein tau accumulate in neurofibrillary tangles in Alzheimer's disease. To investigate the effects of specific phosphorylated tau residues on its function, wild type or phosphomutant tau was expressed in cells. Elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles, without affecting axonal transport kinetics. In contrast, reducing tau phosphorylation enhanced the amount of tau bound to microtubules and inhibited axonal transport of tau. To determine whether differential tau clearance is responsible for the increase in phosphomimic tau, we inhibited autophagy in neurons which resulted in a 3-fold accumulation of phosphomimic tau compared with wild type tau, and endogenous tau was unaffected. In autophagy-deficient mouse embryonic fibroblasts, but not in neurons, proteasomal degradation of phosphomutant tau was also reduced compared with wild type tau. Therefore, autophagic and proteasomal pathways are involved in tau degradation, with autophagy appearing to be the primary route for clearing phosphorylated tau in neurons. Defective autophagy might contribute to the accumulaton of tau in neurodegenerative diseases. PMID:23601672

  17. Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration.

    PubMed

    Salinas-Navarro, Manuel; Alarcón-Martínez, Luis; Valiente-Soriano, Francisco J; Jiménez-López, Manuel; Mayor-Torroglosa, Sergio; Avilés-Trigueros, Marcelino; Villegas-Pérez, María Paz; Vidal-Sanz, Manuel

    2010-01-01

    Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG+ or DTMR+RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR+RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR+RGCs and FG+RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.

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

    SciTech Connect

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

    1989-03-01

    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.

  19. WldS and PGC-1α Regulate Mitochondrial Transport and Oxidation State after Axonal Injury

    PubMed Central

    O'Donnell, Kelley C.; Vargas, Mauricio E.

    2013-01-01

    Mitochondria carry out many of the processes implicated in maintaining axon health or causing axon degeneration, including ATP and reactive oxygen species (ROS) generation, as well as calcium buffering and protease activation. Defects in mitochondrial function and transport are common in axon degeneration, but how changes in specific mitochondrial properties relate to degeneration is not well understood. Using cutaneous sensory neurons of living larval zebrafish as a model, we examined the role of mitochondria in axon degeneration by monitoring mitochondrial morphology, transport, and redox state before and after laser axotomy. Mitochondrial transport terminated locally after injury in wild-type axons, an effect that was moderately attenuated by expressing the axon-protective fusion protein Wallerian degeneration slow (WldS). However, mitochondrial transport arrest eventually occurred in WldS-protected axons, indicating that later in the lag phase, mitochondrial transport is not required for axon protection. By contrast, the redox-sensitive biosensor roGFP2 was rapidly oxidized in the mitochondrial matrix after injury, and WldS expression prevented this effect, suggesting that stabilization of ROS production may mediate axon protection. Overexpression of PGC-1α, a transcriptional coactivator with roles in both mitochondrial biogenesis and ROS detoxification, dramatically increased mitochondrial density, attenuated roGFP2 oxidation, and delayed Wallerian degeneration. Collectively, these results indicate that mitochondrial oxidation state is a more reliable indicator of axon vulnerability to degeneration than mitochondrial motility. PMID:24027278

  20. Kinesin KIF4A transports integrin β1 in developing axons of cortical neurons.

    PubMed

    Heintz, Tristan G; Heller, Janosch P; Zhao, Rongrong; Caceres, Alfredo; Eva, Richard; Fawcett, James W

    2014-11-01

    CNS axons have poor regenerative ability compared to PNS axons, and mature axons regenerate less well than immature embryonic axons. The loss of regenerative ability with maturity is accompanied by the setting up of a selective transport filter in axons, restricting the types of molecule that are present. We confirm that integrins (represented by subunits β1 and α5) are present in early cortical axons in vitro but are excluded from mature axons. Ribosomal protein and L1 show selective axonal transport through association with kinesin kif4A; we have therefore examined the hypothesis that integrin transport might also be in association with kif4A. Kif4A is present in all processes of immature cortical neurons cultured at E18, then downregulated by 14days in vitro, coinciding with the exclusion of integrin from axons. Kif4a co-localises with β1 integrin in vesicles in neurons and non-neuronal cells, and the two molecules co-immunoprecipitate. Knockdown of KIF4A expression with shRNA reduced the level of integrin β1 in axons of developing neurons and reduced neurite elongation on laminin, an integrin-dependent substrate. Overexpression of kif4A triggered apoptosis in neuronal and non-neuronal cells. In mature neurons expression of kif4A-GFP at a modest level did not kill the cells, and the kif4A was detectable in their axons. However this was not accompanied by an increase in integrin β1 axonal transport, suggesting that kif4A is not the only integrin transporter, and that integrin exclusion from axons is controlled by factors other than the kif4A level. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Dynamics of axonal mRNA transport and implications for peripheral nerve regeneration

    PubMed Central

    Yoo, Soonmoon; van Niekerk, Erna A.; Merianda, Tanuja T.; Twiss, Jeffery L.

    2009-01-01

    Locally generating new proteins in subcellular regions provides means to spatially and temporally modify protein content in polarized cells. Recent years have seen resurgence of the concept that axonal processes of neurons can locally synthesize proteins. Experiments from a number of groups have now shown that axonal protein synthesis helps to initiate growth, provides a means to respond to guidance cues, and generates retrograde signaling complexes. Additionally, there is increasing evidence that locally synthesized proteins provide functions beyond injury responses and growth in the mature peripheral nervous system. A key regulatory event in this translational regulation is moving the mRNA templates into the axonal compartment. Transport of mRNAs into axons is a highly regulated and specific process that requires interaction of RNA binding proteins with specific cis-elements or structures within the mRNAs. mRNAs are transported in ribonucleoprotein particles that interact with microtubule motor proteins for long-range axonal transport and likely use microfilaments for short-range movement in the axons. The mature axon is able to recruit mRNAs into translation with injury and possibly other stimuli suggesting that mRNAs can be stored in a dormant state in the distal axon until needed. Axotomy triggers a shift in the populations of mRNAs localized to axons indicating a dynamic regulation of the specificity of the axonal transport machinery. In this review, we discuss how axonal mRNA transport and localization are regulated to achieve specific changes in axonal RNA content in response to axonal stimuli. PMID:19699200

  2. Structural protein transport in elongating motor axons after sciatic nerve crush. Effect of a conditioning lesion.

    PubMed

    McQuarrie, I G

    1986-12-01

    In elongating motor axons of the rat sciatic nerve, the maximum outgrowth rate increased from 4.6 to 5.3 mm/d (5.3-6.1 X 10(-8) m/s) when a testing lesion of spinal nerves L4 and L5 was preceded 2 wk earlier by a conditioning lesion of the sciatic nerve. Axonal outgrowth was examined by measuring the transport of 35[S]methionine-labeled structural proteins (tubulin, actin, and neurofilament triplet) from "parent" axon stumps into "daughter" axon sprouts. Since these proteins are conveyed by the slow component of axonal transport at 1-5 mm/d (1.2-6.0 X 10(-8) m/s), the isotope was injected into the spinal cord 1 wk before the testing lesion. Nerves were removed 8 d after the testing lesion, sectioned into 3-mm segments, and homogenized; soluble proteins were separated by polyacrylamide gel electrophoresis. Fluorographs were used as templates to identify gel segments for removal, solubilization, and liquid scintillation counting. Distributions of mean radioactivity for tubulin, actin, and neurofilament triplet were plotted for animals receiving a conditioning vs sham-conditioning lesion. Greater amounts of tubulin and actin were transported into daughter axons in the conditioned group. Tubulin was mainly increased in axon shafts, whereas actin was mainly increased in axon tips. These findings suggest that the axonal transport of tubulin and actin governs the rate of elongation.

  3. In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS.

    PubMed

    Takihara, Yuji; Inatani, Masaru; Eto, Kei; Inoue, Toshihiro; Kreymerman, Alexander; Miyake, Seiji; Ueno, Shinji; Nagaya, Masatoshi; Nakanishi, Ayami; Iwao, Keiichiro; Takamura, Yoshihiro; Sakamoto, Hirotaka; Satoh, Keita; Kondo, Mineo; Sakamoto, Tatsuya; Goldberg, Jeffrey L; Nabekura, Junichi; Tanihara, Hidenobu

    2015-08-18

    The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23-25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

  4. In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS

    PubMed Central

    Takihara, Yuji; Inatani, Masaru; Eto, Kei; Inoue, Toshihiro; Kreymerman, Alexander; Miyake, Seiji; Ueno, Shinji; Nagaya, Masatoshi; Nakanishi, Ayami; Iwao, Keiichiro; Takamura, Yoshihiro; Sakamoto, Hirotaka; Satoh, Keita; Kondo, Mineo; Sakamoto, Tatsuya; Goldberg, Jeffrey L.; Nabekura, Junichi; Tanihara, Hidenobu

    2015-01-01

    The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23–25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo. PMID:26240337

  5. Axonal transport declines with age in two distinct phases separated by a period of relative stability☆

    PubMed Central

    Milde, Stefan; Adalbert, Robert; Elaman, M. Handan; Coleman, Michael P.

    2015-01-01

    Axonal transport is critical for supplying newly synthesized proteins, organelles, mRNAs, and other cargoes from neuronal cell bodies into axons. Its impairment in many neurodegenerative conditions appears likely to contribute to pathogenesis. Axonal transport also declines during normal aging, but little is known about the timing of these changes, or about the effect of aging on specific cargoes in individual axons. This is important for understanding mechanisms of age-related axon loss and age-related axonal disorders. Here we use fluorescence live imaging of peripheral nerve and central nervous system tissue explants to investigate vesicular and mitochondrial axonal transport. Interestingly, we identify 2 distinct periods of change, 1 period during young adulthood and the other in old age, separated by a relatively stable plateau during most of adult life. We also find that after tibial nerve regeneration, even in old animals, neurons are able to support higher transport rates of each cargo for a prolonged period. Thus, the age-related decline in axonal transport is not an inevitable consequence of either aging neurons or an aging systemic milieu. PMID:25443288

  6. In vivo Imaging of Mitochondrial Transport in Single-Axon Regeneration of Zebrafish Mauthner Cells

    PubMed Central

    Xu, Yang; Chen, Min; Hu, Bingbing; Huang, Rongchen; Hu, Bing

    2017-01-01

    Mitochondrial transport is essential for neuronal function, but the evidence of connections between mitochondrial transport and axon regeneration in the central nervous system (CNS) of living vertebrates remains limited. Here, we developed a novel model to explore mitochondrial transport in a single Mauthner axon (M axon) of zebrafish with non-invasive in vivo imaging. To confirm the feasibility of using this model, we treated labeled zebrafish with nocodazole and demonstrated that it could disrupt mitochondrial transport. We also used two-photon laser axotomy to precisely axotomize M axons and simultaneously recorded their regeneration and the process of mitochondrial transport in living zebrafish larvae. The findings showed that the injured axons with stronger regenerative capability maintain greater mitochondrial motility. Furthermore, to stimulate axon regeneration, treatment with dibutyryl cyclic adenosine monophosphate (db-cAMP) could also augment mitochondrial motility. Taken together, our results provide new evidence that mitochondrial motility is positively correlated with axon regeneration in the living vertebrate CNS. This promising model will be useful for further studies on the interaction between axon regeneration and mitochondrial dynamics, using various genetic and pharmacological techniques. PMID:28174522

  7. [32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo.

    PubMed

    Nixon, R A; Lewis, S E; Mercken, M; Sihag, R K

    1994-11-01

    Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [32P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble 32P-carrier that was axonally transported faster than neurofilaments. 32P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons. 32P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total 32P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [35S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.

  8. The movement of membranous organelles in axons. Electron microscopic identification of anterogradely and retrogradely transported organelles

    PubMed Central

    1980-01-01

    To identify the structures to be rapidly transported through the axons, we developed a new method to permit local cooling of mouse saphenous nerves in situ without exposing them. By this method, both anterograde and retrograde transport were successfully interrupted, while the structural integrity of the nerves was well preserved. Using radioactive tracers, anterogradely transported proteins were shown to accumulate just proximal to the cooled site, and retrogradely transported proteins just distal to the cooled site. Where the anterogradely transported proteins accumulated, the vesiculotubular membranous structures increased in amount inside both myelinated and unmyelinated axons. Such accumulated membranous structures showed a relatively uniform diameter of 50--80 nm, and some of them seemed to be continuous with the axonal smooth endoplasmic reticulum (SER). Thick sections of nerves selectively stained for the axonal membranous structures revealed that the network of the axonal SER was also packed inside axons proximal to the cooled site. In contrast, large membranous bodies of varying sizes accumulated inside axons just distal to the cooled site, where the retrogradely transported proteins accumulated. These bodies were composed mainly of multivesicular bodies and lamellated membranous structures. When horseradish peroxidase was administered in the distal end of the nerve, membranous bodies showing this activity accumulated, together with unstained membranous bodies. Hence, we are led to propose that, besides mitochondria, the membranous components in the axon can be classified into two systems from the viewpoint of axonal transport: "axonal SER and vesiculotubular structures" in the anterograde direction and "large membranous bodies" in the retrograde direction. PMID:6153657

  9. Axonal transport defects are a common phenotype in Drosophila models of ALS

    PubMed Central

    Baldwin, Katie R.; Godena, Vinay K.; Hewitt, Victoria L.; Whitworth, Alexander J.

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons resulting in a catastrophic loss of motor function. Current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Mutations in a large number of genes have now been linked to ALS, including SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and their pathogenic mutations have provided great insights into the underlying disease mechanisms. Defective axonal transport is hypothesized to be a key factor in the selective vulnerability of motor nerves due to their extraordinary length and evidence that ALS occurs as a distal axonopathy. Axonal transport is seen as an early pathogenic event that precedes cell loss and clinical symptoms and so represents an upstream mechanism for therapeutic targeting. Studies have begun to describe the impact of a few pathogenic mutations on axonal transport but a broad survey across a range of models and cargos is warranted. Here, we assessed the axonal transport of different cargos in multiple Drosophila models of ALS. We found that axonal transport defects are common across all models tested, although they often showed a differential effect between mitochondria and vesicle cargos. Motor deficits were also common across the models and generally worsened with age, though surprisingly there was not a clear correlation between the severity of axonal transport defects and motor ability. These results further support defects in axonal transport as a common factor in models of ALS that may contribute to the pathogenic process. PMID:27056981

  10. The ALS disease protein TDP-43 is actively transported in motor neuron axons and regulates axon outgrowth.

    PubMed

    Fallini, Claudia; Bassell, Gary J; Rossoll, Wilfried

    2012-08-15

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease specifically affecting cortical and spinal motor neurons. Cytoplasmic inclusions containing hyperphosphorylated and ubiquitinated TDP-43 are a pathological hallmark of ALS, and mutations in the gene encoding TDP-43 have been directly linked to the development of the disease. TDP-43 is a ubiquitous DNA/RNA-binding protein with a nuclear role in pre-mRNA splicing. However, the selective vulnerability and axonal degeneration of motor neurons in ALS pose the question of whether TDP-43 may have an additional role in the regulation of the cytoplasmic and axonal fate of mRNAs, processes important for neuron function. To investigate this possibility, we have characterized TDP-43 localization and dynamics in primary cultured motor neurons. Using a combination of cell imaging and biochemical techniques, we demonstrate that TDP-43 is localized and actively transported in live motor neuron axons, and that it co-localizes with well-studied axonal mRNA-binding proteins. Expression of the TDP-43 C-terminal fragment led to the formation of hyperphosphorylated and ubiquitinated inclusions in motor neuron cell bodies and neurites, and these inclusions specifically sequestered the mRNA-binding protein HuD. Additionally, we showed that overexpression of full-length or mutant TDP-43 in motor neurons caused a severe impairment in axon outgrowth, which was dependent on the C-terminal protein-interacting domain of TDP-43. Taken together, our results suggest a role of TDP-43 in the regulation of axonal growth, and suggest that impairment in the post-transcriptional regulation of mRNAs in the cytoplasm of motor neurons may be a major factor in the development of ALS.

  11. A close look at axonal transport: Cargos slow down when crossing stationary organelles.

    PubMed

    Che, Daphne L; Chowdary, Praveen D; Cui, Bianxiao

    2016-01-01

    The bidirectional transport of cargos along the thin axon is fundamental for the structure, function and survival of neurons. Defective axonal transport has been linked to the mechanism of neurodegenerative diseases. In this paper, we study the effect of the local axonal environment to cargo transport behavior in neurons. Using dual-color fluorescence imaging in microfluidic neuronal devices, we quantify the transport dynamics of cargos when crossing stationary organelles such as non-moving endosomes and stationary mitochondria in the axon. We show that the axonal cargos tend to slow down, or pause transiently within the vicinity of stationary organelles. The slow-down effect is observed in both retrograde and anterograde transport directions of three different cargos (TrkA, lysosomes and TrkB). Our results agree with the hypothesis that bulky axonal structures can pose as steric hindrance for axonal transport. However, the results do not rule out the possibility that cellular mechanisms causing stationary organelles are also responsible for the delay in moving cargos at the same locations.

  12. There and back again: coordinated transcription, translation and transport in axonal survival and regeneration.

    PubMed

    Tasdemir-Yilmaz, Ozge E; Segal, Rosalind A

    2016-08-01

    Neurons are highly polarized cells with axonal and dendritic projections that extend over long distances. Target-derived neurotrophins provide local axonal cues that function in developing neurons, while physical or chemical injuries to long axons initiate local environmental cues in mature neurons. In both instances initial responses at the location of stimulation or injury must be coordinated with changes in the transcriptional program and subsequent changes in axonal protein content. To achieve this coordination, intracellular signals move 'there and back again' between axons and the nucleus. Here, we review new findings on neuronal responses to growth factors and injury and highlight the coordination of transcription, translation and transport required to mediate communication between axons and cell bodies.

  13. Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal NeuronsV⃞

    PubMed Central

    Kaether, Christoph; Skehel, Paul; Dotti, Carlos G.

    2000-01-01

    Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 μm/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 μm/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier. PMID:10749925

  14. Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits

    PubMed Central

    Yu, Panpan; Lin, Mei-Yao; Chen, Yanmin

    2016-01-01

    Although neuronal regeneration is a highly energy-demanding process, axonal mitochondrial transport progressively declines with maturation. Mature neurons typically fail to regenerate after injury, thus raising a fundamental question as to whether mitochondrial transport is necessary to meet enhanced metabolic requirements during regeneration. Here, we reveal that reduced mitochondrial motility and energy deficits in injured axons are intrinsic mechanisms controlling regrowth in mature neurons. Axotomy induces acute mitochondrial depolarization and ATP depletion in injured axons. Thus, mature neuron-associated increases in mitochondria-anchoring protein syntaphilin (SNPH) and decreases in mitochondrial transport cause local energy deficits. Strikingly, enhancing mitochondrial transport via genetic manipulation facilitates regenerative capacity by replenishing healthy mitochondria in injured axons, thereby rescuing energy deficits. An in vivo sciatic nerve crush study further shows that enhanced mitochondrial transport in snph knockout mice accelerates axon regeneration. Understanding deficits in mitochondrial trafficking and energy supply in injured axons of mature neurons benefits development of new strategies to stimulate axon regeneration. PMID:27268498

  15. Calcium/calmodulin-dependent protein kinase IIalpha in optic axons moves with slow axonal transport and undergoes posttranslational modification.

    PubMed

    Lund, L M; McQuarrie, I G

    2001-12-21

    In neurons, the mRNA for calcium/calmodulin-dependent protein kinase II alpha (CKIIalpha) is known to be targeted to dendrites-where the enzyme is synthesized and supports postsynaptic functions. We are interested in knowing how neuronal proteins enter axons from the nerve cell body, and the mechanism for protein transport to terminals. Because CKIIalpha immunofluorescence can be demonstrated in over 80% of retinal ganglion cells, we asked whether this regulatory protein is being transported into optic axons. Using Sprague-Dawley rats, [(35)S] methionine was injected into the vitreous humor of the eye. Four days later, the optic nerves, tracts, lateral geniculate ganglia, and superior colliculi were removed and processed for 2D-PAGE and Western blotting. Radiolabeled CKIIalpha appears to move with slow component b (SCb) of axonal transport, as is the case in rodent sciatic motor neurons. In addition, the radiolabeled CKIIalpha isoform that enters the optic nerve is found to be 4 kDa heavier (in SDS-PAGE molecular mass) than the isoform in the optic tract, superior colliculus, and lateral geniculate nucleus. This reduction is likely the result of dephosphorylation, which is a mechanism used to regulate the enzyme's activity.

  16. Selective Microtubule-Based Transport of Dendritic Membrane Proteins Arises in Concert with Axon Specification

    PubMed Central

    Petersen, Jennifer D.; Kaech, Stefanie

    2014-01-01

    The polarized distribution of membrane proteins to axonal or somatodendritic neuronal compartments is fundamental to nearly every aspect of neuronal function. The polarity of dendritic proteins depends on selective microtubule-based transport; the vesicles that carry these proteins are transported into dendrites but do not enter the axon. We used live-cell imaging of fluorescently tagged dendritic and axonal proteins combined with immunostaining for initial segment and cytoskeletal markers to evaluate different models of dendrite-selective transport in cultured rat hippocampal neurons. In mature neurons, dendritic vesicles that entered the base of the axon stopped at the proximal edge of the axon initial segment, defined by immunostaining for ankyrinG, rather than moving into the initial segment itself. In contrast, axonal vesicles passed through the initial segment without impediment. During development, dendrite-selective transport was detected shortly after axons formed, several days before initial segment assembly, before the appearance of a dense actin meshwork in the initial segment, and before dendrites acquire microtubules of mixed polarity orientation. Indeed, some elements of selective transport were detected even before axon specification. These findings are inconsistent with models for selective transport that depend on the presence of an F-actin-based cytoplasmic filter in the initial segment or that posit that transport into dendrites is mediated by dyneins translocating along minus-end out microtubules. Instead our results suggest that selective transport involves the coordinated regulation of the different motor proteins that mediate dendritic vesicle transport and that the selectivity of motor-microtubule interactions is one facet of this process. PMID:24647935

  17. Organophosphate Related Alterations in Myelin and Axonal Transport in the Living Mammalian Brain

    DTIC Science & Technology

    2015-10-01

    we evaluated the effects of the commonly used OP- pesticide , chlorpyrifos (CPF) on axonal transport in the brains of living rats using manganese (Mn2...Environ Health Perspect 2004;112(9):950–8. Karlsson JO, Hansson HA, Sjöstrand J. Effect of colchicine on axonal transport and morphology of retinal...Pope CN. Organophosphorus pesticides : do they all have the same mechanism of toxicity? J Toxicol Environ Health B Crit Rev 1999;2:161–81. Pope C

  18. A proposal for a classification of neuropathies according to their axonal transport abnormalities.

    PubMed Central

    Jakobsen, J; Sidenius, P; Braendgaard, H

    1986-01-01

    Recent studies on axonal transport in experimental neuropathy are reviewed and the following combinations of pathological changes and underlying axonal transport abnormalities are proposed for a classification of polyneuropathies. Alterations of the anterograde transport of slow component a(SCa) leads to changes of the dimensions of the axon calibre without the occurrence either of overt neuropathy or fibre loss. Thus damming of SCa in beta,beta'-iminodiproprionitrile (IDPN) intoxication results in axonal swelling in nerve roots whereas decrease of SCa leads to atrophy distal to the swellings in IDPN intoxication and in streptozotocin induced diabetes as well. Decrease in the amount of material conveyed within the anterograde fast component (aFC) leads to acute axonal degeneration including break down of axons and fibre loss. This state occurs in acute hypoglycaemia and in doxorubicin intoxication. The most frequent type of polyneuropathy, namely distal axonopathy with accumulation of axon organelles leading to distal fibre loss, is associated with decrease in amount of the retrograde fast component (rFC). The transport is impaired before the appearance of symptoms and electrophysiological signs of neuropathy develop in the intoxications induced by parabromophenylacetylurea, acrylamide and 2.5 hexanedione, and the severity of neuropathy is proportional to the rFC impairment. PMID:2428941

  19. Optic nerve fast axonal transport abnormalities in primates. Occurrence after short posterior ciliary artery occlusion.

    PubMed

    Radius, R L

    1980-11-01

    Fast axonal transport abnormalities in primate (Aotus trivirgatus) optic nerve were studied in ten eyes at various intervals after occlusion of the lateral short posterior ciliary circulation. Evidence of focal axonal ischemia, as indicated by swelling of mitochondria and dissolution of cytoplasmic detail, was noted as early as one hour after occlusion. Accumulation of mitochondria, microvesicles, and dense bodies, indicating focal interruption of axonal transport mechanisms, was noted in eyes examined at 2, 4, and 6 hours. This accumulation of organelles was limited to the region of the lamina cribrosa. Nerve head abnormalities were not seen in two eyes studied at two weeks.

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

    PubMed

    Yates, Darran M; Manser, Catherine; De Vos, Kurt J; Shaw, Christopher E; McLoughlin, Declan M; Miller, Christopher C J

    2009-04-01

    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.

  1. Axonal transport of proteins. A new view using in vivo covalent labeling

    PubMed Central

    1980-01-01

    The injection of [2,3-3H]N-succinimidyl propionate ([3H]N-SP) into the rat sciatic nerve was used to covalently label both intra- and extra- axonal proteins. While extra-axonal proteins (e.g., myelin proteins) remained in the injection site, the intra-axonal proteins were transported in both the anterograde and retrograde directions. The mobile labeled proteins appeared to move by normal axonal transport processes because: (a) autoradiographic studies showed that they were localized exclusively within the axon at considerable distances from the injection site, (b) specific and identifiable proteins (by SDS gel electrophoresis) moved at expected rates in the anterograde direction, and (c) an entirely different profile of proteins moved in the anterograde vs. retrograde direction. This novel experimental approach to axonal transport, which is independent of de novo protein synthesis, provided a unique view of slow anterograde transport, and particularly of retrograde transport of endogenous proteins. A large quantity of a 68,000 mol wt proteins, moving at approximately 3-6 mm/day, dominated the retograde transport profile. [3H]N-SP, therefore, represents a new and unique "vital stain" which may find many applications in cell biology. PMID:6154709

  2. Vesicular glycolysis provides on-board energy for fast axonal transport.

    PubMed

    Zala, Diana; Hinckelmann, Maria-Victoria; Yu, Hua; Lyra da Cunha, Marcel Menezes; Liot, Géraldine; Cordelières, Fabrice P; Marco, Sergio; Saudou, Frédéric

    2013-01-31

    Fast axonal transport (FAT) requires consistent energy over long distances to fuel the molecular motors that transport vesicles. We demonstrate that glycolysis provides ATP for the FAT of vesicles. Although inhibiting ATP production from mitochondria did not affect vesicles motility, pharmacological or genetic inhibition of the glycolytic enzyme GAPDH reduced transport in cultured neurons and in Drosophila larvae. GAPDH localizes on vesicles via a huntingtin-dependent mechanism and is transported on fast-moving vesicles within axons. Purified motile vesicles showed GAPDH enzymatic activity and produced ATP. Finally, we show that vesicular GAPDH is necessary and sufficient to provide on-board energy for fast vesicular transport. Although detaching GAPDH from vesicles reduced transport, targeting GAPDH to vesicles was sufficient to promote FAT in GAPDH deficient neurons. This specifically localized glycolytic machinery may supply constant energy, independent of mitochondria, for the processive movement of vesicles over long distances in axons. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Impaired retrograde transport of axonal autophagosomes contributes to autophagic stress in Alzheimer’s disease neurons

    PubMed Central

    Tammineni, Prasad; Ye, Xuan; Feng, Tuancheng; Aikal, Daniyal; Cai, Qian

    2017-01-01

    Neurons face unique challenges of transporting nascent autophagic vacuoles (AVs) from distal axons toward the soma, where mature lysosomes are mainly located. Autophagy defects have been linked to Alzheimer’s disease (AD). However, the mechanisms underlying altered autophagy remain unknown. Here, we demonstrate that defective retrograde transport contributes to autophagic stress in AD axons. Amphisomes predominantly accumulate at axonal terminals of mutant hAPP mice and AD patient brains. Amyloid-β (Aβ) oligomers associate with AVs in AD axons and interact with dynein motors. This interaction impairs dynein recruitment to amphisomes through competitive interruption of dynein-Snapin motor-adaptor coupling, thus immobilizing them in distal axons. Consistently, deletion of Snapin in mice causes AD-like axonal autophagic stress, whereas overexpressing Snapin in hAPP neurons reduces autophagic accumulation at presynaptic terminals by enhancing AV retrograde transport. Altogether, our study provides new mechanistic insight into AD-associated autophagic stress, thus establishing a foundation for ameliorating axonal pathology in AD. DOI: http://dx.doi.org/10.7554/eLife.21776.001 PMID:28085665

  4. Target-Derived Neurotrophins Coordinate Transcription and Transport of Bclw to Prevent Axonal Degeneration

    PubMed Central

    Cosker, Katharina E.; Pazyra-Murphy, Maria F.; Fenstermacher, Sara J.

    2013-01-01

    Establishment of neuronal circuitry depends on both formation and refinement of neural connections. During this process, target-derived neurotrophins regulate both transcription and translation to enable selective axon survival or elimination. However, it is not known whether retrograde signaling pathways that control transcription are coordinated with neurotrophin-regulated actions that transpire in the axon. Here we report that target-derived neurotrophins coordinate transcription of the antiapoptotic gene bclw with transport of bclw mRNA to the axon, and thereby prevent axonal degeneration in rat and mouse sensory neurons. We show that neurotrophin stimulation of nerve terminals elicits new bclw transcripts that are immediately transported to the axons and translated into protein. Bclw interacts with Bax and suppresses the caspase6 apoptotic cascade that fosters axonal degeneration. The scope of bclw regulation at the levels of transcription, transport, and translation provides a mechanism whereby sustained neurotrophin stimulation can be integrated over time, so that axonal survival is restricted to neurons connected within a stable circuit. PMID:23516285

  5. Target-derived neurotrophins coordinate transcription and transport of bclw to prevent axonal degeneration.

    PubMed

    Cosker, Katharina E; Pazyra-Murphy, Maria F; Fenstermacher, Sara J; Segal, Rosalind A

    2013-03-20

    Establishment of neuronal circuitry depends on both formation and refinement of neural connections. During this process, target-derived neurotrophins regulate both transcription and translation to enable selective axon survival or elimination. However, it is not known whether retrograde signaling pathways that control transcription are coordinated with neurotrophin-regulated actions that transpire in the axon. Here we report that target-derived neurotrophins coordinate transcription of the antiapoptotic gene bclw with transport of bclw mRNA to the axon, and thereby prevent axonal degeneration in rat and mouse sensory neurons. We show that neurotrophin stimulation of nerve terminals elicits new bclw transcripts that are immediately transported to the axons and translated into protein. Bclw interacts with Bax and suppresses the caspase6 apoptotic cascade that fosters axonal degeneration. The scope of bclw regulation at the levels of transcription, transport, and translation provides a mechanism whereby sustained neurotrophin stimulation can be integrated over time, so that axonal survival is restricted to neurons connected within a stable circuit.

  6. Length of axons expressing the serotonin transporter in orbitofrontal cortex is lower with age in depression.

    PubMed

    Rajkowska, Grazyna; Mahajan, Gouri; Legutko, Beata; Challagundla, Lavanya; Griswold, Michael; Albert, Paul R; Daigle, Mireille; Miguel-Hidalgo, Jose J; Austin, Mark C; Blakely, Randy D; Steffens, David C; Stockmeier, Craig A

    2017-09-17

    Studies of major depressive disorder (MDD) in postmortem brain tissue report enhanced binding to inhibitory serotonin-1A autoreceptors in midbrain dorsal raphe and reductions in length of axons expressing the serotonin transporter (SERT) in dorsolateral prefrontal cortex. The length density of axons expressing SERT in the orbitofrontal cortex (OFC) was determined in 18 subjects with MDD and 17 age-matched control subjects. A monoclonal antibody was used to immunohistochemically label the SERT in fixed sections of OFC. The 3-dimensional length density of SERT-immunoreactive (ir) axons in layer VI of OFC was estimated. The age of subjects with MDD was negatively correlated with SERT axon length (r=-0.77, p<0.0005). The significant effect of age persisted when removing four depressed subjects with an antidepressant medication present at the time of death, or when removing nine depressed subjects that had a recent prescription for an antidepressant medication. Neither gender, tissue pH, postmortem interval, 5-HTTLPR genotype, time in fixative, nor death by suicide had a significant effect on axon length. The age-related decrease in SERT-ir axon length in MDD may reflect pathology of ascending axons passing through deep white matter hyperintensities. Greater length of axons expressing SERT in younger subjects with MDD may result in a significant deficit in serotonin availability in OFC. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Herpes Simplex Virus Capsids Are Transported in Neuronal Axons without an Envelope Containing the Viral Glycoproteins▿ †

    PubMed Central

    Snyder, Aleksandra; Wisner, Todd W.; Johnson, David C.

    2006-01-01

    Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini. PMID:16971450

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

    PubMed Central

    Naftelberg, Shiran; Ast, Gil; Perlson, Eran

    2017-01-01

    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. PMID:28553323

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

    PubMed

    Naftelberg, Shiran; Ast, Gil; Perlson, Eran

    2017-04-01

    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.

  10. The parkinsonian mimetic, MPP+, specifically impairs mitochondrial transport in dopamine axons.

    PubMed

    Kim-Han, Jeong Sook; Antenor-Dorsey, Jo Ann; O'Malley, Karen L

    2011-05-11

    Impaired axonal transport may play a key role in Parkinson's disease. To test this notion, a microchamber system was adapted to segregate axons from cell bodies using green fluorescent protein-labeled mouse dopamine (DA) neurons. Transport was examined in axons challenged with the DA neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+). MPP+ rapidly reduced overall mitochondrial motility in DA axons; among motile mitochondria, anterograde transport was slower yet retrograde transport was increased. Transport effects were specific for DA mitochondria, which were smaller and transported more slowly than their non-DA counterparts. MPP+ did not affect synaptophysin-tagged vesicles or any other measureable moving particle. Toxin effects on DA mitochondria were not dependent upon ATP, calcium, free radical species, JNK, or caspase3/PKC pathways but were completely blocked by the thiol-anti-oxidant N-acetyl-cysteine or membrane-permeable glutathione. Since these drugs also rescued processes from degeneration, these findings emphasize the need to develop therapeutics aimed at axons as well as cell bodies to preserve "normal" circuitry and function as long as possible.

  11. Spreading of α-synuclein in the face of axonal transport deficits in Parkinson's disease: a speculative synthesis.

    PubMed

    Lamberts, Jennifer T; Hildebrandt, Erin N; Brundin, Patrik

    2015-05-01

    Parkinson's disease (PD) is mainly attributed to degeneration of dopamine neurons in the substantia nigra, but its etiopathogenesis also includes impaired protein clearance and axonal transport dysfunction, among others. The spread of α-synuclein (α-syn) aggregates from one neuron to another, in a prion-like manner, is hypothesized to contribute to PD progression. Axonal transport is likely to play a crucial role in this movement of α-syn aggregates between brain regions. At the same time, deficits in axonal transport are suggested to contribute to neuronal failure in PD. In this review, we discuss the apparent contradiction that axonal transport might be essential for disease progression, while dysfunction of axonal transport could simultaneously be a cornerstone of PD pathogenesis. We speculate around models that reconcile how axonal transport can play such a paradoxical role. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Impaired Axonal Transport in Motor Neurons Correlates with Clinical Prion Disease

    PubMed Central

    Ermolayev, Vladimir; Cathomen, Toni; Merk, Julia; Friedrich, Mike; Härtig, Wolfgang; Harms, Gregory S.; Klein, Michael A.; Flechsig, Eckhard

    2009-01-01

    Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally—after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease. PMID:19696919

  13. Molecular characterization of a trafficking organelle: dissecting the axonal paths of calsyntenin-1 transport vesicles.

    PubMed

    Steuble, Martin; Gerrits, Bertran; Ludwig, Alexander; Mateos, José María; Diep, Tu-My; Tagaya, Mitsuo; Stephan, Alexander; Schätzle, Philipp; Kunz, Beat; Streit, Peter; Sonderegger, Peter

    2010-11-01

    Kinesin motors play crucial roles in the delivery of membranous cargo to its destination and thus for the establishment and maintenance of cellular polarization. Recently, calsyntenin-1 was identified as a cargo-docking protein for Kinesin-1-mediated axonal transport of tubulovesicular organelles along axons of central nervous system neurons. To further define the function of calsyntenin-1, we immunoisolated calsyntenin-1 organelles from murine brain homogenates and determined their proteome by MS. We found that calsyntenin-1 organelles are endowed with components of the endosomal trafficking machinery and contained the β-amyloid precursor protein (APP). Detailed biochemical analyses of calsyntenin-1 immunoisolates in conjunction with immunocytochemical colocalization studies with cultured hippocampal neurons, using endosomal marker proteins for distinct subcompartments of the endosomal pathways, indicated that neuronal axons contain at least two distinct, nonoverlapping calsyntenin-1-containing transport packages: one characterized as early-endosomal, APP positive, the other as recycling-endosomal, APP negative. We postulate that calsyntenin-1 acts as a general mediator of anterograde axonal transportation of endosomal vesicles. In this role, calsyntenin-1 may actively contribute to axonal growth and pathfinding in the developing as well as to the maintenance of neuronal polarity in the adult nervous system; further, it may actively contribute to the stabilization of APP during its anterograde axonal trajectory.

  14. Quantitative analysis of axonal transport by using compartmentalized and surface micropatterned culture of neurons.

    PubMed

    Kim, Hyung Joon; Park, Jeong Won; Byun, Jae Hwan; Poon, Wayne W; Cotman, Carl W; Fowlkes, Charless C; Jeon, Noo Li

    2012-06-20

    Mitochondria, synaptic vesicles, and other cytoplasmic constituents have to travel long distance along the axons from cell bodies to nerve terminals. Interruption of this axonal transport may contribute to many neurodegenerative diseases including Alzheimer's disease (AD). It has been recently shown that exposure of cultured neurons to β-amyloid (Aβ) resulted in severe impairment of mitochondrial transport. This Letter describes an integrated microfluidic platform that establishes surface patterned and compartmentalized culture of neurons for studying the effect of Aβ on mitochondria trafficking in full length of axons. We have successfully quantified the trafficking of fluorescently labeled mitochondria in distal and proximal axons using image processing. Selective treatment of Aβ in the somal or axonal compartments resulted in considerable decrease in mitochondria movement in a location dependent manner such that mitochondria trafficking slowed down more significantly proximal to the location of Aβ exposure. Furthermore, this result suggests a promising application of microfluidic technology for investigating the dysfunction of axonal transport related to neurodegenerative diseases.

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

    SciTech Connect

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

    1989-12-01

    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.

  16. Redistribution of proteins of fast axonal transport following administration of beta,beta'-iminodipropionitrile: a quantitative autoradiographic study

    PubMed Central

    1982-01-01

    Beta,beta'-iminodipropionitrile (IDPN) produces a rearrangement of axoplasmic organelles with displacement of microtubules, smooth endoplasmic reticulum, and mitochondria toward the center and of neurofilaments toward the periphery of the axon, whereas the rate of the fast component of axonal transport is unchanged. Separation of microtubules and neurofilaments makes the IDPN axons an excellent model for study of the role of these two organelles in axonal transport. The cross-sectional distribution of [3H]-labeled proteins moving with the front of the fast transport was analyzed by quantitative electron microscopic autoradiography in sciatic nerves of IDPN-treated and control rats, 6 h after injection of a 1:1 mixture of [3H]-proline and [3H]-lysine into lumbar ventral horns. In IDPN axons most of the transported [3H] proteins were located in the central region with microtubules, smooth endoplasmic reticulum and mitochondria, whereas few or none were in the periphery with neurofilaments. In control axons the [3H]-labeled proteins were uniformly distributed within the axoplasm. It is concluded that in fast axonal transport: (a) neurofilaments play no primary role; (b) the normal architecture of the axonal cytoskeleton and the normal cross-sectional distribution of transported materials are not indispensable for the maintenance of a normal rate of transport. The present findings are consistent with the models of fast transport that envision microtubules as the key organelles in providing directionality and propulsive force to the fast component of axonal transport. PMID:6183280

  17. Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon.

    PubMed

    Galbraith, J A; Reese, T S; Schlief, M L; Gallant, P E

    1999-09-28

    A major issue in the slow transport of cytoskeletal proteins is the form in which they are transported. We have investigated the possibility that unpolymerized as well as polymerized cytoskeletal proteins can be actively transported in axons. We report the active transport of highly diffusible tubulin oligomers, as well as transport of the less diffusible neurofilament polymers. After injection into the squid giant axon, tubulin was transported in an anterograde direction at an average rate of 2.3 mm/day, whereas neurofilament was moved at 1.1 mm/day. Addition of the metabolic poisons cyanide or dinitrophenol reduced the active transport of both proteins to less than 10% of control values, whereas disruption of microtubules by treatment of the axon with cold in the presence of nocodazole reduced transport of both proteins to approximately 20% of control levels. Passive diffusion of these proteins occurred in parallel with transport. The diffusion coefficient of the moving tubulin in axoplasm was 8.6 micrometer(2)/s compared with only 0.43 micrometer(2)/s for neurofilament. These results suggest that the tubulin was transported in the unpolymerized state and that the neurofilament was transported in the polymerized state by an energy-dependent nocodazole/cold-sensitive transport mechanism.

  18. Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon

    PubMed Central

    Galbraith, James A.; Reese, Thomas S.; Schlief, Michelle L.; Gallant, Paul E.

    1999-01-01

    A major issue in the slow transport of cytoskeletal proteins is the form in which they are transported. We have investigated the possibility that unpolymerized as well as polymerized cytoskeletal proteins can be actively transported in axons. We report the active transport of highly diffusible tubulin oligomers, as well as transport of the less diffusible neurofilament polymers. After injection into the squid giant axon, tubulin was transported in an anterograde direction at an average rate of 2.3 mm/day, whereas neurofilament was moved at 1.1 mm/day. Addition of the metabolic poisons cyanide or dinitrophenol reduced the active transport of both proteins to less than 10% of control values, whereas disruption of microtubules by treatment of the axon with cold in the presence of nocodazole reduced transport of both proteins to ≈20% of control levels. Passive diffusion of these proteins occurred in parallel with transport. The diffusion coefficient of the moving tubulin in axoplasm was 8.6 μm2/s compared with only 0.43 μm2/s for neurofilament. These results suggest that the tubulin was transported in the unpolymerized state and that the neurofilament was transported in the polymerized state by an energy-dependent nocodazole/cold-sensitive transport mechanism. PMID:10500221

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

    PubMed Central

    2010-01-01

    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 transport holds great promise

  20. Kinesin-1–syntaphilin coupling mediates activity-dependent regulation of axonal mitochondrial transport

    PubMed Central

    Chen, Yanmin

    2013-01-01

    Axonal mitochondria are recruited to synaptic terminals in response to neuronal activity, but the mechanisms underlying activity-dependent regulation of mitochondrial transport are largely unknown. In this paper, using genetic mouse model combined with live imaging, we demonstrate that syntaphilin (SNPH) mediates the activity-dependent immobilization of axonal mitochondria through binding to KIF5. In vitro analysis showed that the KIF5–SNPH coupling inhibited the motor adenosine triphosphatase. Neuronal activity further recruited SNPH to axonal mitochondria. This motor-docking interplay was induced by Ca2+ and synaptic activity and was necessary to establish an appropriate balance between motile and stationary axonal mitochondria. Deleting snph abolished the activity-dependent immobilization of axonal mitochondria. We propose an “Engine-Switch and Brake” model, in which SNPH acts both as an engine off switch by sensing mitochondrial Rho guanosine triphosphatase-Ca2+ and as a brake by anchoring mitochondria to the microtubule track. Altogether, our study provides new mechanistic insight into the molecular interplay between motor and docking proteins, which arrests axonal mitochondrial transport in response to changes in neuronal activity. PMID:23857772

  1. CAR-associated vesicular transport of an adenovirus in motor neuron axons.

    PubMed

    Salinas, Sara; Bilsland, Lynsey G; Henaff, Daniel; Weston, Anne E; Keriel, Anne; Schiavo, Giampietro; Kremer, Eric J

    2009-05-01

    Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.

  2. Ultramicroscopy Reveals Axonal Transport Impairments in Cortical Motor Neurons at Prion Disease

    PubMed Central

    Ermolayev, Vladimir; Friedrich, Mike; Nozadze, Revaz; Cathomen, Toni; Klein, Michael A.; Harms, Gregory S.; Flechsig, Eckhard

    2009-01-01

    Abstract The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice. In combination with tracing as a functional assay for axonal transport, retrogradely labeled descending motor neurons were visualized with >4 mm penetration depth. The analysis of the motor cortex shortly before the onset of clinical prion disease revealed that >80% neurons have functional impairments in axonal transport. Our study provides evidence that prion disease is associated with severe axonal transport defects in the cortical motor neurons and suggests a novel mechanism for prion-mediated neurodegeneration. PMID:19383482

  3. Mycalolide B dissociates dynactin and abolishes retrograde axonal transport of dense-core vesicles.

    PubMed

    Cavolo, Samantha L; Zhou, Chaoming; Ketcham, Stephanie A; Suzuki, Matthew M; Ukalovic, Kresimir; Silverman, Michael A; Schroer, Trina A; Levitan, Edwin S

    2015-07-15

    Axonal transport is critical for maintaining synaptic transmission. Of interest, anterograde and retrograde axonal transport appear to be interdependent, as perturbing one directional motor often impairs movement in the opposite direction. Here live imaging of Drosophila and hippocampal neuron dense-core vesicles (DCVs) containing a neuropeptide or brain-derived neurotrophic factor shows that the F-actin depolymerizing macrolide toxin mycalolide B (MB) rapidly and selectively abolishes retrograde, but not anterograde, transport in the axon and the nerve terminal. Latrunculin A does not mimic MB, demonstrating that F-actin depolymerization is not responsible for unidirectional transport inhibition. Given that dynactin initiates retrograde transport and that amino acid sequences implicated in macrolide toxin binding are found in the dynactin component actin-related protein 1, we examined dynactin integrity. Remarkably, cell extract and purified protein experiments show that MB induces disassembly of the dynactin complex. Thus imaging selective retrograde transport inhibition led to the discovery of a small-molecule dynactin disruptor. The rapid unidirectional inhibition by MB suggests that dynactin is absolutely required for retrograde DCV transport but does not directly facilitate ongoing anterograde DCV transport in the axon or nerve terminal. More generally, MB's effects bolster the conclusion that anterograde and retrograde axonal transport are not necessarily interdependent. © 2015 Cavolo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  4. A simple method for imaging axonal transport in aging neurons using the adult Drosophila wing.

    PubMed

    Vagnoni, Alessio; Bullock, Simon L

    2016-09-01

    There is growing interest in the link between axonal cargo transport and age-associated neuronal dysfunction. The study of axonal transport in neurons of adult animals requires intravital or ex vivo imaging approaches, which are laborious and expensive in vertebrate models. We describe simple, noninvasive procedures for imaging cargo motility within axons using sensory neurons of the translucent Drosophila wing. A key aspect is a method for mounting the intact fly that allows detailed imaging of transport in wing neurons. Coupled with existing genetic tools in Drosophila, this is a tractable system for studying axonal transport over the life span of an animal and thus for characterization of the relationship between cargo dynamics, neuronal aging and disease. Preparation of a sample for imaging takes ∼5 min, with transport typically filmed for 2-3 min per wing. We also document procedures for the quantification of transport parameters from the acquired images and describe how the protocol can be adapted to study other cell biological processes in aging neurons.

  5. A simple method for imaging axonal transport in ageing neurons using the adult Drosophila wing

    PubMed Central

    Vagnoni, Alessio; Bullock, Simon L.

    2016-01-01

    There is growing interest in the link between axonal cargo transport and age-associated neuronal dysfunction. Studying axonal transport in neurons of adult animals requires intravital or ex vivo imaging approaches, which are laborious and expensive in vertebrate models. We describe simple, non-invasive procedures for imaging cargo motility within axons using sensory neurons of the translucent Drosophila wing. A key aspect is a method for mounting the intact fly that allows detailed imaging of transport in wing neurons. Coupled with existing genetic tools in Drosophila, this is a tractable system for studying axonal transport over the lifespan of an animal and thus for characterising the relationship between cargo dynamics, neuronal ageing and disease. Preparation of a sample for imaging takes approximately 5 minutes, with transport typically filmed for 2–3 minutes per wing. We also document procedures for quantifying transport parameters from the acquired images and describe how the protocol can be adapted to study other cell biological processes in ageing neurons. PMID:27560175

  6. Kinesin I transports tetramerized Kv3 channels through the axon initial segment via direct binding.

    PubMed

    Xu, Mingxuan; Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2010-11-24

    Precise targeting of various voltage-gated ion channels to proper membrane domains is crucial for their distinct roles in neuronal excitability and synaptic transmission. How each channel protein is transported within the cytoplasm is poorly understood. Here, we report that KIF5/kinesin I transports Kv3.1 voltage-gated K(+) (Kv) channels through the axon initial segment (AIS) via direct binding. First, we have identified a novel interaction between Kv3.1 and KIF5, confirmed by immunoprecipitation from mouse brain lysates and by pull-down assays with exogenously expressed proteins. The interaction is mediated by a direct binding between the Kv3.1 N-terminal T1 domain and a conserved region in KIF5 tail domains, in which proper T1 tetramerization is crucial. Overexpression of this region of KIF5B markedly reduces axonal levels of Kv3.1bHA. In mature hippocampal neurons, endogenous Kv3.1b and KIF5 colocalize. Suppressing the endogenous KIF5B level by RNA interference significantly reduces the Kv3.1b axonal level. Furthermore, mutating the Zn(2+)-binding site within T1 markedly decreases channel axonal targeting and forward trafficking, likely through disrupting T1 tetramerization and hence eliminating the binding to KIF5 tail. The mutation also alters channel activity. Interestingly, coexpression of the YFP (yellow fluorescent protein)-tagged KIF5B assists dendritic Kv3.1a and even mutants with a faulty axonal targeting motif to penetrate the AIS. Finally, fluorescently tagged Kv3.1 channels colocalize and comove with KIF5B along axons revealed by two-color time-lapse imaging. Our findings suggest that the binding to KIF5 ensures properly assembled and functioning Kv3.1 channels to be transported into axons.

  7. Tracking Quantum-Dot labeled neurotropic factors transport along primary neuronal axons in compartmental microfluidic chambers.

    PubMed

    Gluska, Shani; Chein, Michael; Rotem, Nimrod; Ionescu, Ariel; Perlson, Eran

    2016-01-01

    Neurons are highly polarized cells, with very long axons. Neurotrophic factors like the neuronal growth factor (NGF) are secreted from neuronal targets to promote neuron survival and proper function. These neurotrophic factors must undergo retrograde axonal transport towards the cell body, wherein they initiate signaling pathways important for neurons' various functions and overall health. This process of long-distance axonal signaling is conducted by the dynein motor protein, which transmits signaling endosomes of ligand-receptor complexes retrogradely along microtubule tracks. Here we describe step by step the use of polydimethylsiloxane (PDMS) compartmentalized microfluidic chambers for tracking axonal transport of trophic factors, with a focus on labeled NGF. We describe in detail how to fabricate the molds, assemble the PDMS platform, plate neurons and image, as well as analyze NGF transport along the axon. This method is useful for studying molecular communication mechanisms within the neuron's different compartments as well as between the neuron and its diverse microenvironments, both in health and under pathological conditions.

  8. Relay of retrograde synaptogenic signals through axonal transport of BMP receptors

    PubMed Central

    Smith, Rebecca B.; Machamer, James B.; Kim, Nam Chul; Hays, Thomas S.; Marqués, Guillermo

    2012-01-01

    Summary Neuronal function depends on the retrograde relay of growth and survival signals from the synaptic terminal, where the neuron interacts with its targets, to the nucleus, where gene transcription is regulated. Activation of the Bone Morphogenetic Protein (BMP) pathway at the Drosophila larval neuromuscular junction results in nuclear accumulation of the phosphorylated form of the transcription factor Mad in the motoneuron nucleus. This in turn regulates transcription of genes that control synaptic growth. How BMP signaling at the synaptic terminal is relayed to the cell body and nucleus of the motoneuron to regulate transcription is unknown. We show that the BMP receptors are endocytosed at the synaptic terminal and transported retrogradely along the axon. Furthermore, this transport is dependent on BMP pathway activity, as it decreases in the absence of ligand or receptors. We further demonstrate that receptor traffic is severely impaired when Dynein motors are inhibited, a condition that has previously been shown to block BMP pathway activation. In contrast to these results, we find no evidence for transport of phosphorylated Mad along the axons, and axonal traffic of Mad is not affected in mutants defective in BMP signaling or retrograde transport. These data support a model in which complexes of activated BMP receptors are actively transported along the axon towards the cell body to relay the synaptogenic signal, and that phosphorylated Mad at the synaptic terminal and cell body represent two distinct molecular populations. PMID:22573823

  9. Tau reduction prevents Aβ-induced axonal transport deficits by blocking activation of GSK3β

    PubMed Central

    Xu, Jordan C.; Fomenko, Vira; Miyamoto, Takashi; Suberbielle, Elsa; Knox, Joseph A.; Ho, Kaitlyn; Kim, Daniel H.; Yu, Gui-Qiu

    2015-01-01

    Axonal transport deficits in Alzheimer’s disease (AD) are attributed to amyloid β (Aβ) peptides and pathological forms of the microtubule-associated protein tau. Genetic ablation of tau prevents neuronal overexcitation and axonal transport deficits caused by recombinant Aβ oligomers. Relevance of these findings to naturally secreted Aβ and mechanisms underlying tau’s enabling effect are unknown. Here we demonstrate deficits in anterograde axonal transport of mitochondria in primary neurons from transgenic mice expressing familial AD-linked forms of human amyloid precursor protein. We show that these deficits depend on Aβ1–42 production and are prevented by tau reduction. The copathogenic effect of tau did not depend on its microtubule binding, interactions with Fyn, or potential role in neuronal development. Inhibition of neuronal activity, N-methyl-d-aspartate receptor function, or glycogen synthase kinase 3β (GSK3β) activity or expression also abolished Aβ-induced transport deficits. Tau ablation prevented Aβ-induced GSK3β activation. Thus, tau allows Aβ oligomers to inhibit axonal transport through activation of GSK3β, possibly by facilitating aberrant neuronal activity. PMID:25963821

  10. Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila

    PubMed Central

    Reis, Gerald F.; Yang, Ge; Szpankowski, Lukasz; Weaver, Carole; Shah, Sameer B.; Robinson, John T.; Hays, Thomas S.; Danuser, Gaudenz; Goldstein, Lawrence S. B.

    2012-01-01

    Bidirectional axonal transport driven by kinesin and dynein along microtubules is critical to neuronal viability and function. To evaluate axonal transport mechanisms, we developed a high-resolution imaging system to track the movement of amyloid precursor protein (APP) vesicles in Drosophila segmental nerve axons. Computational analyses of a large number of moving vesicles in defined genetic backgrounds with partial reduction or overexpression of motor proteins enabled us to test with high precision existing and new models of motor activity and coordination in vivo. We discovered several previously unknown features of vesicle movement, including a surprising dependence of anterograde APP vesicle movement velocity on the amount of kinesin-1. This finding is largely incompatible with the biophysical properties of kinesin-1 derived from in vitro analyses. Our data also suggest kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and their direction and velocity are controlled at least in part by dynein intermediate chain. PMID:22398725

  11. HIV Glycoprotein Gp120 Impairs Fast Axonal Transport by Activating Tak1 Signaling Pathways

    PubMed Central

    Berth, Sarah H.; Mesnard-Hoaglin, Nichole; Wang, Bin; Kim, Hajwa; Song, Yuyu; Sapar, Maria; Morfini, Gerardo

    2016-01-01

    Sensory neuropathies are the most common neurological complication of HIV. Of these, distal sensory polyneuropathy (DSP) is directly caused by HIV infection and characterized by length-dependent axonal degeneration of dorsal root ganglion (DRG) neurons. Mechanisms for axonal degeneration in DSP remain unclear, but recent experiments revealed that the HIV glycoprotein gp120 is internalized and localized within axons of DRG neurons. Based on these findings, we investigated whether intra-axonal gp120 might impair fast axonal transport (FAT), a cellular process critical for appropriate maintenance of the axonal compartment. Significantly, we found that gp120 severely impaired both anterograde and retrograde FAT. Providing a mechanistic basis for these effects, pharmacological experiments revealed an involvement of various phosphotransferases in this toxic effect, including members of mitogen-activated protein kinase pathways (Tak-1, p38, and c-Jun N-terminal Kinase (JNK)), inhibitor of kappa-B-kinase 2 (IKK2), and PP1. Biochemical experiments and axonal outgrowth assays in cell lines and primary cultures extended these findings. Impairments in neurite outgrowth in DRG neurons by gp120 were rescued using a Tak-1 inhibitor, implicating a Tak-1 mitogen-activated protein kinase pathway in gp120 neurotoxicity. Taken together, these observations indicate that kinase-based impairments in FAT represent a novel mechanism underlying gp120 neurotoxicity consistent with the dying-back degeneration seen in DSP. Targeting gp120-based impairments in FAT with specific kinase inhibitors might provide a novel therapeutic strategy to prevent axonal degeneration in DSP. PMID:27872270

  12. Neurotoxic mechanisms of paclitaxel are local to the distal axon and independent of transport defects.

    PubMed

    Gornstein, Erica L; Schwarz, Thomas L

    2017-02-01

    Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of paclitaxel and other chemotherapeutic agents. Paclitaxel binds and stabilizes microtubules, but the cellular mechanisms that underlie paclitaxel's neurotoxic effects are not well understood. We therefore used primary cultures of adult murine dorsal root ganglion neurons, the cell type affected in patients, to examine leading hypotheses to explain paclitaxel neurotoxicity. We address the role of microtubule hyperstabilization and its downstream effects. Paclitaxel administered at 10-50nM for 1-3days induced retraction bulbs at the tips of axons and arrested axon growth without triggering axon fragmentation or cell death. By correlating the toxic effects and microtubule stabilizing activity of structurally different microtubule stabilizing compounds, we confirmed that microtubule hyperstabilization, rather than an off-target effect, is the likely primary cause of paclitaxel neurotoxicity. We examined potential downstream consequences of microtubule hyperstabilization and found that changes in levels of tubulin posttranslational modifications, although present after paclitaxel exposure, are not implicated in the paclitaxel neurotoxicity we observed in the cultures. Additionally, defects in axonal transport were not implicated as an early, causative mechanism of paclitaxel's toxic effects on dorsal root ganglion neurons. By using microfluidic chambers to selectively treat different parts of the axon with paclitaxel, we found that the distal axon was primarily vulnerable to paclitaxel, indicating that paclitaxel acts directly on the distal axon to induce degenerative effects. Together, our findings point to local effects of microtubule hyperstabilization on the distal-most portion of the axon as an early mediator of paclitaxel neurotoxicity. Because sensory neurons have a unique and ongoing requirement for distal growth in order to reinnervate the epidermis as it turns over, we propose

  13. Axonal Segregation and Role of the Vesicular Glutamate Transporter VGLUT3 in Serotonin Neurons

    PubMed Central

    Voisin, Aurore N.; Mnie-Filali, Ouissame; Giguère, Nicolas; Fortin, Guillaume M.; Vigneault, Erika; El Mestikawy, Salah; Descarries, Laurent; Trudeau, Louis-Éric

    2016-01-01

    A subset of monoamine neurons releases glutamate as a cotransmitter due to presence of the vesicular glutamate transporters VGLUT2 or VGLUT3. In addition to mediating vesicular loading of glutamate, it has been proposed that VGLUT3 enhances serotonin (5-HT) vesicular loading by the vesicular monoamine transporter (VMAT2) in 5-HT neurons. In dopamine (DA) neurons, glutamate appears to be released from specialized subsets of terminals and it may play a developmental role, promoting neuronal growth and survival. The hypothesis of a similar developmental role and axonal localization of glutamate co-release in 5-HT neurons has not been directly examined. Using postnatal mouse raphe neurons in culture, we first observed that in contrast to 5-HT itself, other phenotypic markers of 5-HT axon terminals such as the 5-HT reuptake transporter (SERT) show a more restricted localization in the axonal arborization. Interestingly, only a subset of SERT- and 5-HT-positive axonal varicosities expressed VGLUT3, with SERT and VGLUT3 being mostly segregated. Using VGLUT3 knockout mice, we found that deletion of this transporter leads to reduced survival of 5-HT neurons in vitro and also decreased the density of 5-HT-immunoreactivity in terminals in the dorsal striatum and dorsal part of the hippocampus in the intact brain. Our results demonstrate that raphe 5-HT neurons express SERT and VGLUT3 mainly in segregated axon terminals and that VGLUT3 regulates the vulnerability of these neurons and the neurochemical identity of their axonal domain, offering new perspectives on the functional connectivity of a cell population involved in anxiety disorders and depression. PMID:27147980

  14. Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function.

    PubMed

    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

    2014-04-01

    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.

  15. Glycine Transporter-1 Inhibition Promotes Striatal Axon Sprouting via NMDA Receptors in Dopamine Neurons

    PubMed Central

    Castagna, Candace; Mrejeru, Ana; Lizardi-Ortiz, José E.; Klein, Zoe; Lindsley, Craig W.

    2013-01-01

    NMDA receptor activity is involved in shaping synaptic connections throughout development and adulthood. We recently reported that brief activation of NMDA receptors on cultured ventral midbrain dopamine neurons enhanced their axon growth rate and induced axonal branching. To test whether this mechanism was relevant to axon regrowth in adult animals, we examined the reinnervation of dorsal striatum following nigral dopamine neuron loss induced by unilateral intrastriatal injections of the toxin 6-hydroxydopamine. We used a pharmacological approach to enhance NMDA receptor-dependent signaling by treatment with an inhibitor of glycine transporter-1 that elevates levels of extracellular glycine, a coagonist required for NMDA receptor activation. All mice displayed sprouting of dopaminergic axons from spared fibers in the ventral striatum to the denervated dorsal striatum at 7 weeks post-lesion, but the reinnervation in mice treated for 4 weeks with glycine uptake inhibitor was approximately twice as dense as in untreated mice. The treated mice also displayed higher levels of striatal dopamine and a complete recovery from lateralization in a test of sensorimotor behavior. We confirmed that the actions of glycine uptake inhibition on reinnervation and behavioral recovery required NMDA receptors in dopamine neurons using targeted deletion of the NR1 NMDA receptor subunit in dopamine neurons. Glycine transport inhibitors promote functionally relevant sprouting of surviving dopamine axons and could provide clinical treatment for disorders such as Parkinson's disease. PMID:24133278

  16. Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis.

    PubMed

    Kreutzer, Mihaela; Seehusen, Frauke; Kreutzer, Robert; Pringproa, Kidsadagorn; Kummerfeld, Maren; Claus, Peter; Deschl, Ulrich; Kalkul, Arno; Beineke, Andreas; Baumgärtner, Wolfgang; Ulrich, Reiner

    2012-07-01

    Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease characterized by myelin and axonal pathology. In a viral model of MS, we tested whether axonopathy initiation and development are based on an impaired transport of neurofilaments. Spinal cords of Theiler's murine encephalomyelitis virus (TMEV)-infected and mock-infected mice and TMEV infected neuroblastoma N1E-115 cells were analyzed by microarray analysis, light microscopy and electron and laser confocal microscopy. In vivo axonal accumulation of non-phosphorylated neurofilaments after TMEV infection revealed a temporal development caused by the impairments of the axonal traffic consisting of the downregulation of kinesin family member 5A, dynein cytoplasmic heavy chain 1, tau-1 and β-tubulin III expression. In addition, alterations of the protein metabolism were also noticed. In vitro, the TMEV-infected N1E-115 cells developed tandem-repeated swellings similar to in vivo alterations. Furthermore, the hypothesis of an underlying axonal self-destruction program involving nicotinamide adenine dinucleotide depletion was supported by molecular findings. The obtained data indicate that neurofilament accumulation in TME is mainly the result of dysregulation of their axonal transport machinery and impairment of neurofilament phosphorylation and protein metabolism. The present findings allow a more precise understanding of the complex interactions responsible for initiation and development of axonopathies in inflammatory degenerative diseases.

  17. Dominant-negative myosin Va impairs retrograde but not anterograde axonal transport of large dense core vesicles.

    PubMed

    Bittins, Claudia Margarethe; Eichler, Tilo Wolf; Hammer, John A; Gerdes, Hans-Hermann

    2010-04-01

    Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport.

  18. UV Irradiation Accelerates Amyloid Precursor Protein (APP) Processing and Disrupts APP Axonal Transport

    PubMed Central

    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.

    2014-01-01

    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

  19. Effect of MSH/ACTH peptides on fast axonal transport in intact and regenerating sciatic nerves

    SciTech Connect

    Crescitelli, L.A.

    1985-01-01

    Fast axonal transport was examined in intact rats treated with ACTH 4-10 or ACTH 4-9 (ORG 2766), hypophysectomized rats, adrenalectomized rats, and in ACTH 4-10 treated rats with crushed regenerating sciatic nerves by injecting /sup 3/H-leucine into the ventral horn region of the spinal cord. The distance traveled by the transported activity along the sciatic nerve and the rate of fast axonal transport were not significantly altered as a result of treatment with ACTH 4-10, ACTH 4-9 (ORG 2766), hypophysectomy, or adrenalectomy. Treatment with ACTH 4-9 (ORG 2766) at concentrations of 1 ..mu..g/Kg /day and 10 ..mu..g/Kg/day caused significant reductions (62% and 64% respectively) in the crest height of the fast axonal transport curve as compared to 0.9% saline treated control animals. No significant differences were found in comparing the distance, rate, slope, or crest height of ACTH 4-10 treated animals with crushed regenerating (7 or 14d) sciatic nerves to control animals. In the group of animals in days, the amount of radiolabeled activity was significantly increased in the ACTH 4-10 treated animals as compared to control animals. The results indicate that during regeneration the peptide acts to prolong the initially high levels of synthetic activity which occur in regenerating axons.

  20. Berberine Attenuates Axonal Transport Impairment and Axonopathy Induced by Calyculin A in N2a Cells

    PubMed Central

    Abid, Morad Dirhem Naji; Yan, Huanhuan; Huang, Hao; Wan, Limin; Feng, Zuohua; Chen, Juan

    2014-01-01

    Berberine is a primary component of the most functional extracts of Coptidis rhizome used in traditional Chinese medicine for centuries. Recent reports indicate that Berberine has the potential to prevent and treat Alzheimer's disease (AD). The previous studies reported that Calyculin A (CA) impaired the axonal transport in neuroblastoma-2a (N2a) cells. Berberine attenuated tau hyperphosphorylation and cytotoxicity induced by CA. Our study aimed at investigating the effects of Berberine on the axonal transport impairment induced by CA in N2a cells. The results showed that Berberine could protect the cell from CA -induced toxicity in metabolism and viability, as well as hyperphosphorylation of tau and neurofilaments (NFs). Furthermore, Berberine could reverse CA-induced axonal transport impairment significantly. Berberine also partially reversed the phosphorylation of the catalytic subunit of PP-2A at Tyrosine 307, a crucial site negatively regulating the activity of PP-2A, and reduced the levels of malondialdehyde and the activity of superoxide dismutase, markers of oxidative stress, induced by CA. The present work for the first time demonstrates that Berberine may play a role in protecting against CA-induced axonal transport impairment by modulating the activity of PP-2A and oxidative stress. Our findings also suggest that Berberine may be a potential therapeutic drug for AD. PMID:24713870

  1. Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity.

    PubMed

    Zhang, Peisu; Abdelmohsen, Kotb; Liu, Yong; Tominaga-Yamanaka, Kumiko; Yoon, Je-Hyun; Ioannis, Grammatikakis; Martindale, Jennifer L; Zhang, Yongqing; Becker, Kevin G; Yang, In Hong; Gorospe, Myriam; Mattson, Mark P

    2015-11-20

    Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a non-telomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release.

  2. Axonal transport of calmodulin: a physiologic approach to identification of long-term associations between proteins

    PubMed Central

    1981-01-01

    Calmodulin is a soluble, heat-stable protein which has been shown to modulate both membrane-bound and soluble enzymes, but relatively little has been known about the in vivo associations of calmodulin. A 17,000- dalton heat-stable protein was found to move in axonal transport in the guinea pig visual system with the proteins of slow component b (SCb; 2 mm/d) along with actin and the bulk of the soluble proteins of the axon. Co-electrophoresis of purified calmodulin and radioactively labeled SCb proteins in two dimensional polyacrylamide gel electrophoresis (PAGE) demonstrated the identity of the heat-stable SCb protein and calmodulin on the basis of pI, molecular weight, and anomalous migration in the presence of Ca2+-chelating agents. No proteins co-migrating with calmodulin in two-dimensional PAGE could be detected among the proteins of slow component a (SCa; 0.3 mm/d, microtubules and neurofilaments) or fast component (FC; 250 mm/d, membrane-associated proteins). We conclude that calmodulin is transported solely as part of the SCb complex of proteins, the axoplasmic matrix. Calmodulin moves in axonal transport independent of the movements of microtubules (SCa) and membranes (FC), which suggests that the interactions of calmodulin with these structures may represent a transient interaction between groups of proteins moving in axonal transport at different rates. Axonal transport has been shown to be an effective tool for the demonstration of long-term in vivo protein associations. PMID:6166619

  3. Loss of Fractalkine Signaling Exacerbates Axon Transport Dysfunction in a Chronic Model of Glaucoma

    PubMed Central

    Breen, Kevin T.; Anderson, Sarah R.; Steele, Michael R.; Calkins, David J.; Bosco, Alejandra; Vetter, Monica L.

    2016-01-01

    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 proinflammatory

  4. Non-Cell-Autonomous Regulation of Retrograde Motoneuronal Axonal Transport in an SBMA Mouse Model

    PubMed Central

    Halievski, Katherine; Kemp, Michael Q.; Breedlove, S. Marc; Miller, Kyle E.

    2016-01-01

    Abstract Defects in axonal transport are seen in motoneuronal diseases, but how that impairment comes about is not well understood. In spinal bulbar muscular atrophy (SBMA), a disorder linked to a CAG/polyglutamine repeat expansion in the androgen receptor (AR) gene, the disease-causing AR disrupts axonal transport by acting in both a cell-autonomous fashion in the motoneurons themselves, and in a non-cell-autonomous fashion in muscle. The non-cell-autonomous mechanism is suggested by data from a unique “myogenic” transgenic (TG) mouse model in which an AR transgene expressed exclusively in skeletal muscle fibers triggers an androgen-dependent SBMA phenotype, including defects in retrograde transport. However, motoneurons in this TG model retain the endogenous AR gene, leaving open the possibility that impairments in transport in this model also depend on ARs in the motoneurons themselves. To test whether non-cell-autonomous mechanisms alone can perturb retrograde transport, we generated male TG mice in which the endogenous AR allele has the testicular feminization mutation (Tfm) and, consequently, is nonfunctional. Males carrying the Tfm allele alone show no deficits in motor function or axonal transport, with or without testosterone treatment. However, when Tfm males carrying the myogenic transgene (Tfm/TG) are treated with testosterone, they develop impaired motor function and defects in retrograde transport, having fewer retrogradely labeled motoneurons and deficits in endosomal flux based on time-lapse video microscopy of living axons. These findings demonstrate that non-cell-autonomous disease mechanisms originating in muscle are sufficient to induce defects in retrograde transport in motoneurons. PMID:27517091

  5. Non-Cell-Autonomous Regulation of Retrograde Motoneuronal Axonal Transport in an SBMA Mouse Model.

    PubMed

    Halievski, Katherine; Kemp, Michael Q; Breedlove, S Marc; Miller, Kyle E; Jordan, Cynthia L

    2016-01-01

    Defects in axonal transport are seen in motoneuronal diseases, but how that impairment comes about is not well understood. In spinal bulbar muscular atrophy (SBMA), a disorder linked to a CAG/polyglutamine repeat expansion in the androgen receptor (AR) gene, the disease-causing AR disrupts axonal transport by acting in both a cell-autonomous fashion in the motoneurons themselves, and in a non-cell-autonomous fashion in muscle. The non-cell-autonomous mechanism is suggested by data from a unique "myogenic" transgenic (TG) mouse model in which an AR transgene expressed exclusively in skeletal muscle fibers triggers an androgen-dependent SBMA phenotype, including defects in retrograde transport. However, motoneurons in this TG model retain the endogenous AR gene, leaving open the possibility that impairments in transport in this model also depend on ARs in the motoneurons themselves. To test whether non-cell-autonomous mechanisms alone can perturb retrograde transport, we generated male TG mice in which the endogenous AR allele has the testicular feminization mutation (Tfm) and, consequently, is nonfunctional. Males carrying the Tfm allele alone show no deficits in motor function or axonal transport, with or without testosterone treatment. However, when Tfm males carrying the myogenic transgene (Tfm/TG) are treated with testosterone, they develop impaired motor function and defects in retrograde transport, having fewer retrogradely labeled motoneurons and deficits in endosomal flux based on time-lapse video microscopy of living axons. These findings demonstrate that non-cell-autonomous disease mechanisms originating in muscle are sufficient to induce defects in retrograde transport in motoneurons.

  6. Neuronal overexpression of APPL, the Drosophila homologue of the amyloid precursor protein (APP), disrupts axonal transport.

    PubMed

    Torroja, L; Chu, H; Kotovsky, I; White, K

    1999-05-06

    The two pathological hallmarks of Alzheimer's disease, amyloid plaques and neurofibrillary tangles, involve two apparently unrelated proteins, the amyloid precursor protein (APP) and Tau. Although it is known that aberrant processing of APP is associated with Alzheimer's disease, the definitive role of APP in neurons is not yet clear. Tau regulates microtubule stabilization and assembly in axons and is, thus, an essential component of the microtubule-associated organelle transport machinery. Although several groups have reported physical interaction between APP and Tau, and induction of Tau phosphorylation by APP and beta-amyloid peptide, the functional connection between APP and Tau is unclear. To explore the possibility that the functions of these two proteins may somehow converge on the same cellular process, we overexpressed APPL, the Drosophila homologue of APP, along with Tau in Drosophila neurons. Panneural coexpression of APPL and Tau resulted in adults that, upon eclosion, failed to expand wings and harden the cuticle, which is suggestive of neuroendocrine dysfunction. We analyzed axonal transport when Tau and APPL were coexpressed and found that transport of axonal cargo was disrupted, as evidenced by increased retention of synaptic proteins in axons and scarcity of neuropeptide-containing vesicles in the distal processes of peptidergic neurons. In an independent approach, we demonstrated genetic interaction and phenotypic similarity between APPL overexpression and mutations in the Kinesin heavy chain (Khc) gene, the product of which is a motor for anterograde vesicle trafficking.

  7. ER transport on actin filaments in squid giant axon: implications for signal transduction at synapse.

    PubMed

    Langford, G M

    1999-12-01

    The smooth endoplasmic reticulum (S-ER) is transported on actin filaments in the giant axon of the squid. The identity of the myosin motors that transport S-ER in the squid giant axon has been determined. Our recent studies have shown that the motor for movement of S-ER vesicles on actin filaments is Myosin-V (1). These findings grew out of a series of studies that began with the initial observation that vesicles in the giant axon of the squid move on both microtubules and actin filaments (2). These initial studies documented the ability of individual vesicles to move from microtubules to actin filaments and led to the development of the dual filament model of vesicle transport (3, 4). The model proposes that long-range movement of vesicles occurs on microtubules and short-range movement on actin filaments. S-ER vesicles were identified as the major population of vesicles in the axon that use myosin-V for movement on actin filaments. The S-ER is the primary site of calcium storage, and it regulates the local cytosolic calcium concentration. Calcium release from the S-ER in neurons couples electrical excitation to signal transduction cascades. The signaling cascades triggered by the release of calcium from S-ER in dendritic spines are postulated to initiate the cellular mechanisms that lead to learning and memory.

  8. Defective axonal transport: A common pathological mechanism in inherited and acquired peripheral neuropathies.

    PubMed

    Prior, Robert; Van Helleputte, Lawrence; Benoy, Veronick; Van Den Bosch, Ludo

    2017-09-01

    Peripheral neuropathies are characterized by a progressive and length-dependent loss of peripheral nerve function. This can be caused either by genetic defects, classified as 'inherited peripheral neuropathies', or they can be acquired throughout life. In that case, the disease is caused by various insults such as toxins and mechanical injuries, or it can arise secondary to medical conditions such as metabolic disorders, nutritional deficiencies, inflammation and infections. Peripheral neuropathies are not only very heterogeneous in etiology, but also in their pathology and clinical presentation. A commonality amongst all peripheral neuropathies is that no pharmacological disease-modifying therapies currently exist that can reverse or cure these diseases. Moreover, the length-dependent nature of the disease, affecting the longest nerves at the most distal sites, suggests an important role for disturbances in axonal transport, directly or indirectly linked to alterations in the cytoskeleton. In this review, we will give a systematic overview of the main arguments for the involvement of axonal transport defects in both inherited and acquired peripheral neuropathies. In addition, we will discuss the possible therapeutic strategies that can potentially counteract these disturbances, as this particular pathway might be a promising strategy to find a cure. Since counteracting axonal transport defects could limit the axonal degeneration and could be a driving force for neuronal regeneration, the benefits might be twofold. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Assessment of retinal ganglion cell damage in glaucomatous optic neuropathy: Axon transport, injury and soma loss.

    PubMed

    Nuschke, Andrea C; Farrell, Spring R; Levesque, Julie M; Chauhan, Balwantray C

    2015-12-01

    Glaucoma is a disease characterized by progressive axonal pathology and death of retinal ganglion cells (RGCs), which causes structural changes in the optic nerve head and irreversible vision loss. Several experimental models of glaucomatous optic neuropathy (GON) have been developed, primarily in non-human primates and, more recently and commonly, in rodents. These models provide important research tools to study the mechanisms underlying glaucomatous damage. Moreover, experimental GON provides the ability to quantify and monitor risk factors leading to RGC loss such as the level of intraocular pressure, axonal health and the RGC population. Using these experimental models we are able to gain a better understanding of GON, which allows for the development of potential neuroprotective strategies. Here we review the advantages and disadvantages of the relevant and most often utilized methods for evaluating axonal degeneration and RGC loss in GON. Axonal pathology in GON includes functional disruption of axonal transport (AT) and structural degeneration. Horseradish peroxidase (HRP), rhodamine-B-isothiocyanate (RITC) and cholera toxin-B (CTB) fluorescent conjugates have proven to be effective reporters of AT. Also, immunohistochemistry (IHC) for endogenous AT-associated proteins is often used as an indicator of AT function. Similarly, structural degeneration of axons in GON can be investigated via changes in the activity and expression of key axonal enzymes and structural proteins. Assessment of axonal degeneration can be measured by direct quantification of axons, qualitative grading, or a combination of both methods. RGC loss is the most frequently quantified variable in studies of experimental GON. Retrograde tracers can be used to quantify RGC populations in rodents via application to the superior colliculus (SC). In addition, in situ IHC for RGC-specific proteins is a common method of RGC quantification used in many studies. Recently, transgenic mouse models

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

    PubMed

    Naftelberg, Shiran; Abramovitch, Ziv; Gluska, Shani; Yannai, Sivan; Joshi, Yuvraj; Donyo, Maya; Ben-Yaakov, Keren; Gradus, Tal; Zonszain, Jonathan; Farhy, Chen; Ashery-Padan, Ruth; Perlson, Eran; Ast, Gil

    2016-12-01

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

    PubMed Central

    Naftelberg, Shiran; Abramovitch, Ziv; Gluska, Shani; Yannai, Sivan; Joshi, Yuvraj; Donyo, Maya; Ben-Yaakov, Keren; Gradus, Tal; Zonszain, Jonathan; Farhy, Chen; Ashery-Padan, Ruth

    2016-01-01

    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. PMID:27997532

  12. Semaphorin3A-induced axonal transport mediated through phosphorylation of Axin-1 by GSK3β.

    PubMed

    Hida, Tomonobu; Nakamura, Fumio; Usui, Hiroshi; Takeuchi, Kan; Yamashita, Naoya; Goshima, Yoshio

    2015-02-19

    The establishment of neuronal polarity is necessary for proper neuronal wiring. Semaphorin3A (Sema3A), originally identified as a repulsive axon guidance molecule, exerts a wide variety of biological functions through signaling pathways including sequential phosphorylation of collapsin response mediator protein by cyclin-dependent kinase-5 (Cdk5) and glycogen synthase kinase-3β (GSK3β). Sema3A acts on its receptor neuropilin-1 to regulate axonal transport. To delineate mechanism by which Sema3A induces axonal transport, we investigate whether GSK3β is involved in mediating Sema3A-induced axonal transport. 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, an inhibitor of GSK3β, suppressed Sema3A-induced antero- and retrograde axonal transport. Introduction of either GSK3β mutants, GSK3β-L128A or K85M, suppressed Sema3A-induced axonal transport. On the other hand, introduction of GSK3β-R96A did not affect the Sema3A effect, suggesting that unprimed substrates are primarily involved in Sema3A-induced axonal transport. Overexpression of a partial fragment of frequently rearranged in advanced T-cell lymphomas 1 (FRATtide), which interferes the interaction between GSK3β and Axis inhibitor-1 (Axin-1), also suppressed Sema3A-induced transport. siRNA knockdown of Axin-1, an unprimed substrate of GSK3β, suppressed Sema3A-induced antero- and retrograde axonal transport. These results indicate that GSK3β and Axin-1 are involved in Sema3A-induced bidirectional axonal transport. This finding should provide a clue for understanding of mechanisms of a wide variety of biological activities of Sema3A.

  13. Ankyrin-G Directly Binds to Kinesin-1 to Transport Voltage-Gated Na+ Channels into Axons

    PubMed Central

    Barry, Joshua; Gu, Yuanzheng; Jukkola, Peter; O'Neill, Brian; Gu, Howard; Mohler, Peter J.; Rajamani, Keerthi Thirtamara; Gu, Chen

    2014-01-01

    Action potentials propagating along axons require the activation of voltage-gated Na+ (Nav) channels. How Nav channels are transported into axons is unknown. Here we show KIF5/kinesin-1 directly binds to ankyrin-G (AnkG) to transport Nav channels into axons. KIF5 and Nav1.2 channels bind to multiple sites in the AnkG N-terminal domain that contains 24 ankyrin repeats. Disrupting AnkG-KIF5 binding with siRNA or dominant-negative constructs markedly reduced Nav channel levels at the axon initial segment (AIS) and along entire axons, thereby decreasing action potential firing. Live-cell imaging showed that fluorescently-tagged AnkG or Nav1.2 co-transported with KIF5 along axons. Deleting AnkG in vivo or virus-mediated expression of a dominant-negative KIF5 construct specifically decreased the axonal level of Nav but not Kv1.2 channels in the mouse cerebellum. These results indicate AnkG functions as an adaptor to link Nav channels to KIF5 during axonal transport, before anchoring them to the AIS and nodes of Ranvier. PMID:24412576

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

    PubMed Central

    Steuble, Martin; Diep, Tu-My; Schätzle, Philipp; Ludwig, Alexander; Tagaya, Mitsuo; Kunz, Beat; Sonderegger, Peter

    2012-01-01

    Summary 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. PMID:23213470

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

    PubMed

    Le, Michelle H; Weissmiller, April M; Monte, Louise; Lin, Po Han; Hexom, Tia C; Natera, Orlangie; Wu, Chengbiao; Rissman, Robert A

    2016-01-01

    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.

  16. Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

    PubMed Central

    Koch, J C; Bitow, F; Haack, J; d'Hedouville, Z; Zhang, J-N; Tönges, L; Michel, U; Oliveira, L M A; Jovin, T M; Liman, J; Tatenhorst, L; Bähr, M; Lingor, P

    2015-01-01

    Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered. PMID:26158517

  17. Adeno-Associated Virus Serotypes 1, 8, and 9 Share Conserved Mechanisms for Anterograde and Retrograde Axonal Transport

    PubMed Central

    Castle, Michael J.; Gershenson, Zachary T.; Giles, April R.; Holzbaur, Erika L.F.

    2014-01-01

    Abstract Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane

  18. Axonal-Transport-Mediated Gene Transduction in the Interior of Rat Bone

    PubMed Central

    Okabayashi, Toshitaka; Nakanishi, Kuniaki; Tsuchihara, Toyokazu; Arino, Hiroshi; Yoshihara, Yasuo; Tominaga, Susumu; Uenoyama, Maki; Suzuki, Shinya; Asagiri, Masataka; Nemoto, Koichi

    2010-01-01

    Background Gene transduction has been considered advantageous for the sustained delivery of proteins to specific target tissues. However, in the case of hard tissues, such as bone, local gene delivery remains problematic owing to anatomical accessibility limitations of the target sites. Methodology/Principal Findings Here, we evaluated the feasibility of exogenous gene transduction in the interior of bone via axonal transport following intramuscular administration of a nonviral vector. A high expression level of the transduced gene was achieved in the tibia ipsilateral to the injected tibialis anterior muscle, as well as in the ipsilateral sciatic nerve and dorsal root ganglia. In sciatic transection rats, the gene expression level was significantly lowered in bone. Conclusions/Significance These results suggest that axonal transport is critical for gene transduction. Our study may provide a basis for developing therapeutic methods for efficient gene delivery into hard tissues. PMID:20927397

  19. Studies of Kinesins and Axonal Transport in a Mouse Model of NF1

    DTIC Science & Technology

    2008-03-01

    synaptotagmin (rabbit polyclonal antibody, Abcam ab10104; 1 :500 dilution). Blots were blocked with 3% BSA in PBS. Primary antibody incubations were done...against tubulin (a control antigen) and synaptotagmin and syntaxin - 1 (synaptic proteins that are transported anterograde down the axon...syntaxin 1 , and synaptotagmin . Intensity of staining with these synaptic markers in the segment immediately proximal to the ligature in wild-type

  20. Tau isoforms imbalance impairs the axonal transport of the amyloid precursor protein in human neurons.

    PubMed

    Lacovich, Valentina; Espindola, Sonia L; Alloatti, Matías; Pozo Devoto, Victorio; Cromberg, Lucas; Čarná, Mária; Forte, Giancarlo; Gallo, Jean-Marc; Bruno, Luciana; Stokin, Gorazd B; Avale, M Elena; Falzone, Tomás L

    2016-11-11

    Tau, as a microtubule-associated protein, participates in key neuronal functions such as the regulation of microtubule dynamics, axonal transport and neurite outgrowth. Alternative splicing of exon 10 in the tau primary transcript gives rise to protein isoforms with three (3R) or four (4R) microtubule binding repeats. While tau isoforms are balanced in the normal adult human brain, imbalances in 3R:4R ratio have been tightly associated to the pathogenesis of several neurodegenerative disorders, yet the underlying molecular mechanisms remain elusive. Several studies exploiting tau overexpression and/or mutations suggested that perturbations in tau metabolism impair axonal transport. Nevertheless, no physiological model has yet demonstrated the consequences of altering the endogenous relative content of tau isoforms over axonal transport regulation. Here we addressed this question using a trans-splicing strategy that allows modulating tau exon 10 inclusion/exclusion in differentiated human-derived neurons. Upon changes in 3R:4R tau relative content neurons showed no morphological changes, but live imaging studies revealed that the dynamics of the amyloid precursor protein (APP) were significantly impaired. Single trajectories analyses of the moving vesicles showed that predominance of 3R tau favored the anterograde movement of APP-vesicles, increasing anterograde run lengths and reducing retrograde runs and segmental velocities. Contrarely, the imbalance towards the 4R isoform promoted a retrograde bias by a significant reduction of anterograde velocities. These findings suggest that changes in 3R:4R tau ratio has an impact on the regulation of axonal transport and specifically in APP dynamics, which might link tau isoforms imbalances with APP abnormal metabolism in neurodegenerative processes.

  1. Tau Isoforms Imbalance Impairs the Axonal Transport of the Amyloid Precursor Protein in Human Neurons.

    PubMed

    Lacovich, Valentina; Espindola, Sonia L; Alloatti, Matías; Pozo Devoto, Victorio; Cromberg, Lucas E; Čarná, Mária E; Forte, Giancarlo; Gallo, Jean-Marc; Bruno, Luciana; Stokin, Gorazd B; Avale, M Elena; Falzone, Tomás L

    2017-01-04

    Tau, as a microtubule (MT)-associated protein, participates in key neuronal functions such as the regulation of MT dynamics, axonal transport, and neurite outgrowth. Alternative splicing of exon 10 in the tau primary transcript gives rise to protein isoforms with three (3R) or four (4R) MT binding repeats. Although tau isoforms are balanced in the normal adult human brain, imbalances in 3R:4R ratio have been tightly associated with the pathogenesis of several neurodegenerative disorders, yet the underlying molecular mechanisms remain elusive. Several studies exploiting tau overexpression and/or mutations suggested that perturbations in tau metabolism impair axonal transport. Nevertheless, no physiological model has yet demonstrated the consequences of altering the endogenous relative content of tau isoforms over axonal transport regulation. Here, we addressed this issue using a trans-splicing strategy that allows modulating tau exon 10 inclusion/exclusion in differentiated human-derived neurons. Upon changes in 3R:4R tau relative content, neurons showed no morphological changes, but live imaging studies revealed that the dynamics of the amyloid precursor protein (APP) were significantly impaired. Single trajectory analyses of the moving vesicles showed that predominance of 3R tau favored the anterograde movement of APP vesicles, increasing anterograde run lengths and reducing retrograde runs and segmental velocities. Conversely, the imbalance toward the 4R isoform promoted a retrograde bias by a significant reduction of anterograde velocities. These findings suggest that changes in 3R:4R tau ratio has an impact on the regulation of axonal transport and specifically in APP dynamics, which might link tau isoform imbalances with APP abnormal metabolism in neurodegenerative processes.

  2. Release of axonally transported material from an in vitro amphibian sciatic nerve preparation

    SciTech Connect

    Snyder, R.E.

    1988-04-01

    The rapid axonal transport of a pulse of (35S)methionine-labelled material was used to study the release of transported material from amphibian nerve maintained in vitro. Following creation of a moving pulse of activity in a dorsal root ganglion-sciatic nerve preparation, the ganglion was removed and the nerve placed in a three-compartment tray, the section of nerve in the middle compartment containing no truncated branches (unbranched section). All three compartments were filled with a saline solution that in some studies contained nonradioactive methionine (1.0 mmol/L). Analysis of studies in which nonradioactive methionine was absent revealed that labelled material appeared in the bathing solution of the end compartments that contained truncated branches, but not in the solution of the middle (unbranched) compartment. The quantity of label released in the branched compartments was approximately 6% of that remaining in the corresponding section of nerve following an 18-20 h incubation period. However, when nonradioactive methionine was present, all compartments showed an additional activity in the bathing solution of approximately 10% of that remaining in the nerve. In another study in which a position-sensitive detector of ionizing radiation was used to monitor progress of the pulse, it was found that activity did not enter the bathing solution of a compartment prior to the pulse of activity. It is concluded that in the absence of methionine from the bathing solution, axonally transported material is released only from regions of nerve that contain severed axons; however, the presence of methionine allows transported material to be released from nerve containing intact axons. Ultrafiltration studies and thin-layer chromatography revealed the majority of material released to be of low-molecular weight (less than 30,000 daltons) and not free (35S)methionine.

  3. Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta'-iminodipropionitrile-intoxicated axon as a model system

    PubMed Central

    1985-01-01

    Axons from rats treated with the neurotoxic agent beta,beta'- iminodipropionitrile (IDPN) were examined by quick-freeze, deep-etch electron microscopy. Microtubules formed bundles in the central region of the axons, whereas neurofilaments were segregated to the periphery. Most membrane-bounded organelles, presumably including those involved in rapid axonal transport, were associated with the microtubule domain. The high resolution provided by quick-freeze, deep-etch electron microscopy revealed that the microtubules were coated with an extensive network of fine strands that served both to cross-link the microtubules and to interconnect them with the membrane-bounded organelles. The strands were decorated with granular materials and were irregular in dimension. They appeared either singly or as an extensive anastomosing network in fresh axons. The microtubule-associated strands were observed in fresh, saponin-extracted, or aldehyde-fixed tissue. To explore further the identity of the microtubule-associated strands, microtubules purified from brain tissue and containing the high molecular weight microtubule-associated proteins MAP 1 and MAP 2 were examined by quick-freeze, deep-etch electron microscopy. The purified microtubules were connected by a network of strands quite similar in appearance to those observed in the IDPN axons. Control microtubule preparations consisting only of tubulin and lacking the MAPs were devoid of associated strands. To learn which of the MAPs were present in the microtubule bundles in the axon, sections of axons from IDPN- treated rats were examined by immunofluorescence microscopy using antibodies to MAP 1A, MAP 1B, MAP 2, and tubulin. Anti-MAP 2 staining was only marginally detectable in the IDPN-treated axons, consistent with earlier observations. Anti-MAP 1A and anti-MAP 1B brightly stained the IDPN-treated axons, with the staining exclusively limited to the microtubule domains. Furthermore, thin section-immunoelectron microscopy

  4. K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

    PubMed Central

    1995-01-01

    We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter

  5. Kinesin-2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila.

    PubMed

    Baqri, Rehan; Charan, Rakshita; Schimmelpfeng, Kristina; Chavan, Swati; Ray, Krishanu

    2006-03-01

    Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre- and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule-dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin-2 motor subunit Klp64D and separate siRNA-mediated knock-outs of all the three kinesin-2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin-2 subunits but not with each other. Altogether, these suggest that kinesin-2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors. Copyright 2006 Wiley Periodicals, Inc.

  6. Axonal Transport and Neurodegeneration: How Marine Drugs Can Be Used for the Development of Therapeutics

    PubMed Central

    White, Joseph A.; Banerjee, Rupkatha; Gunawardena, Shermali

    2016-01-01

    Unlike virtually any other cells in the human body, neurons are tasked with the unique problem of transporting important factors from sites of synthesis at the cell bodies, across enormous distances, along narrow-caliber projections, to distally located nerve terminals in order to maintain cell viability. As a result, axonal transport is a highly regulated process whereby necessary cargoes of all types are packaged and shipped from one end of the neuron to the other. Interruptions in this finely tuned transport have been linked to many neurodegenerative disorders including Alzheimer’s (AD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) suggesting that this pathway is likely perturbed early in disease progression. Therefore, developing therapeutics targeted at modifying transport defects could potentially avert disease progression. In this review, we examine a variety of potential compounds identified from marine aquatic species that affect the axonal transport pathway. These compounds have been shown to function in microtubule (MT) assembly and maintenance, motor protein control, and in the regulation of protein degradation pathways, such as the autophagy-lysosome processes, which are defective in many degenerative diseases. Therefore, marine compounds have great potential in developing effective treatment strategies aimed at early defects which, over time, will restore transport and prevent cell death. PMID:27213408

  7. Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

    PubMed Central

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

    2015-01-01

    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

  8. Miro's N-terminal GTPase domain is required for transport of mitochondria into axons and dendrites.

    PubMed

    Babic, Milos; Russo, Gary J; Wellington, Andrea J; Sangston, Ryan M; Gonzalez, Migdalia; Zinsmaier, Konrad E

    2015-04-08

    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.

  9. Diisopropylfluorophosphate Impairs the Transport of Membrane-Bound Organelles in Rat Cortical Axons.

    PubMed

    Gao, Jie; Naughton, Sean X; Wulff, Heike; Singh, Vikrant; Beck, Wayne D; Magrane, Jordi; Thomas, Bobby; Kaidery, Navneet Ammal; Hernandez, Caterina M; Terry, Alvin V

    2016-03-01

    The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OP-related neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons. Here, using a time-lapse imaging technique, we evaluated the OP-nerve agent diisopropylfluorophosphate (DFP) across a wide range of concentrations (subnanomolar to micromolar) for effects on fast axonal transport of membrane-bound organelles (MBOs) that contain the amyloid precursor protein (APP) tagged with the fluorescent marker Dendra2 (APPDendra2). Both 1 and 24 hours of exposure to DFP and a positive control compound, colchicine, resulted in a decrease in the velocity of anterograde and retrograde movements of MBOs and an increase in the number of stationary MBOs. These effects occurred at picomolar (100 pM) to low nanomolar (0.1 nM) concentrations that were not associated with compromised cell viability or cytoskeletal damage. Moreover, the effects of DFP on axonal transport occurred at concentrations that did not inhibit AChE activity, and they were not blocked by cholinergic receptor antagonists. Given the fundamental importance of axonal transport to neuronal function, these observations may explain some of the long-term neurologic deficits that have been observed in humans who have been exposed to OPs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  10. Diisopropylfluorophosphate Impairs the Transport of Membrane-Bound Organelles in Rat Cortical Axons

    PubMed Central

    Gao, Jie; Naughton, Sean X.; Wulff, Heike; Singh, Vikrant; Beck, Wayne D.; Magrane, Jordi; Thomas, Bobby; Kaidery, Navneet Ammal; Hernandez, Caterina M.

    2016-01-01

    The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OP-related neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons. Here, using a time-lapse imaging technique, we evaluated the OP-nerve agent diisopropylfluorophosphate (DFP) across a wide range of concentrations (subnanomolar to micromolar) for effects on fast axonal transport of membrane-bound organelles (MBOs) that contain the amyloid precursor protein (APP) tagged with the fluorescent marker Dendra2 (APPDendra2). Both 1 and 24 hours of exposure to DFP and a positive control compound, colchicine, resulted in a decrease in the velocity of anterograde and retrograde movements of MBOs and an increase in the number of stationary MBOs. These effects occurred at picomolar (100 pM) to low nanomolar (0.1 nM) concentrations that were not associated with compromised cell viability or cytoskeletal damage. Moreover, the effects of DFP on axonal transport occurred at concentrations that did not inhibit AChE activity, and they were not blocked by cholinergic receptor antagonists. Given the fundamental importance of axonal transport to neuronal function, these observations may explain some of the long-term neurologic deficits that have been observed in humans who have been exposed to OPs. PMID:26718240

  11. Pressure-induced optic nerve axonal transport interruption in cat eyes.

    PubMed

    Radius, R L; Bade, B

    1981-12-01

    After intravitreal injection of tritiated leucine, optic nerve axonal transport was studied in 30 cat eyes by tissue radioautography. Twenty-five experimental eyes were examined after four hours of acute pressure elevation with perfusion pressures maintained at 20 to 70 mm Hg. In five control specimens, intraocular pressures were maintained at 10 mm Hg for the four-hour interval. The extent of leucine accumulation, as seen by radioautographs, was inversely proportional to the perfusion pressure. Accumulation was limited to the region fo the lamina cribrosa. The anatomic distribution and pressure response of this transport interruption were similar to those seen in primate eyes studied under similar conditions.

  12. Extracellular Tau Oligomers Induce Invasion of Endogenous Tau into the Somatodendritic Compartment and Axonal Transport Dysfunction

    PubMed Central

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

    2017-01-01

    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

  13. Extracellular Tau Oligomers Induce Invasion of Endogenous Tau into the Somatodendritic Compartment and Axonal Transport Dysfunction.

    PubMed

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

    2017-01-01

    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.

  14. Reversible disruption of dynactin 1-mediated retrograde axonal transport in polyglutamine-induced motor neuron degeneration.

    PubMed

    Katsuno, Masahisa; Adachi, Hiroaki; Minamiyama, Makoto; Waza, Masahiro; Tokui, Keisuke; Banno, Haruhiko; Suzuki, Keisuke; Onoda, Yu; Tanaka, Fumiaki; Doyu, Manabu; Sobue, Gen

    2006-11-22

    Spinal and bulbar muscular atrophy (SBMA) is a hereditary neurodegenerative disease caused by an expansion of a trinucleotide CAG repeat encoding the polyglutamine tract in the androgen receptor (AR) gene. To elucidate the pathogenesis of polyglutamine-mediated motor neuron dysfunction, we investigated histopathological and biological alterations in a transgenic mouse model of SBMA carrying human pathogenic AR. In affected mice, neurofilaments and synaptophysin accumulated at the distal motor axon. A similar intramuscular accumulation of neurofilament was detected in the skeletal muscle of SBMA patients. Fluoro-gold labeling and sciatic nerve ligation demonstrated an impaired retrograde axonal transport in the transgenic mice. The mRNA level of dynactin 1, an axon motor for retrograde transport, was significantly reduced in the SBMA mice resulting from pathogenic AR-induced transcriptional dysregulation. These pathological events were observed before the onset of neurological symptoms, but were reversed by castration, which prevents nuclear accumulation of pathogenic AR. Overexpression of dynactin 1 mitigated neuronal toxicity of the pathogenic AR in a cell culture model of SBMA. These observations indicate that polyglutamine-dependent transcriptional dysregulation of dynactin 1 plays a crucial role in the reversible neuronal dysfunction in the early stage of SBMA.

  15. Botulinum Neurotoxins A and E Undergo Retrograde Axonal Transport in Primary Motor Neurons

    PubMed Central

    Manich, Maria; Bercsenyi, Kinga; Menendez, Guillermo; Rossetto, Ornella; Caleo, Matteo; Schiavo, Giampietro

    2012-01-01

    The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes

  16. In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

    PubMed

    Moya, K L; Confaloni, A M; Allinquant, B

    1994-11-01

    We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

  17. The pseudorabies virus protein, pUL56, enhances virus dissemination and virulence but is dispensable for axonal transport

    PubMed Central

    Daniel, Gina R.; Sollars, Patricia J.; Pickard, Gary E.; Smith, Gregory A.

    2015-01-01

    Neurotropic herpesviruses exit the peripheral nervous system and return to exposed body surfaces following reactivation from latency. The pUS9 protein is a critical viral effector of the anterograde axonal transport that underlies this process. We recently reported that while pUS9 increases the frequency of sorting of newly assembled pseudorabies virus particles to axons from the neural soma during egress, subsequent axonal transport of individual virus particles occurs with wild-type kinetics in the absence of the protein. Here, we examine the role of a related pseudorabies virus protein, pUL56, during neuronal infection. The findings indicate that pUL56 is a virulence factor that supports virus dissemination in vivo, yet along with pUS9, is dispensable for axonal transport. PMID:26655235

  18. A PIK3C3–Ankyrin-B–Dynactin pathway promotes axonal growth and multiorganelle transport

    PubMed Central

    Lorenzo, Damaris Nadia; Badea, Alexandra; Davis, Jonathan; Hostettler, Janell; He, Jiang; Zhong, Guisheng; Zhuang, Xiaowei

    2014-01-01

    Axon growth requires long-range transport of organelles, but how these cargoes recruit their motors and how their traffic is regulated are not fully resolved. In this paper, we identify a new pathway based on the class III PI3-kinase (PIK3C3), ankyrin-B (AnkB), and dynactin, which promotes fast axonal transport of synaptic vesicles, mitochondria, endosomes, and lysosomes. We show that dynactin associates with cargo through AnkB interactions with both the dynactin subunit p62 and phosphatidylinositol 3-phosphate (PtdIns(3)P) lipids generated by PIK3C3. AnkB knockout resulted in shortened axon tracts and marked reduction in membrane association of dynactin and dynein, whereas it did not affect the organization of spectrin–actin axonal rings imaged by 3D-STORM. Loss of AnkB or of its linkages to either p62 or PtdIns(3)P or loss of PIK3C3 all impaired organelle transport and particularly retrograde transport in hippocampal neurons. Our results establish new functional relationships between PIK3C3, dynactin, and AnkB that together promote axonal transport of organelles and are required for normal axon length. PMID:25533844

  19. Excitotoxic oligodendrocyte death and axonal damage induced by glutamate transporter inhibition.

    PubMed

    Domercq, María; Etxebarria, Estibaliz; Pérez-Samartín, Alberto; Matute, Carlos

    2005-10-01

    Glutamate uptake is crucial to terminate glutamate signaling and to prevent excitotoxicity. The present study describes the expression of functional glutamate transporters GLAST and GLT-1 in oligodendrocytes by means of electrophysiology, uptake assays, and immunocytochemistry. Inhibition of glutamate uptake, both in oligodendrocyte cultures and in isolated optic nerves, increases glutamate levels and causes oligodendrocyte excitotoxicity, which is prevented by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptor antagonists. Furthermore, glutamate transporter inhibitors or antisense oligonucleotides applied onto the optic nerve in vivo lead to oligodendroglial loss, massive demyelination, and severe axonal damage. Overall, these results demonstrate that the integrity of oligodendrocytes and white matter depends on proper glutamate transporter function. Deregulated transporter activity may contribute to acute and chronic white matter damage.

  20. Soluble N-terminal fragment of mutant Huntingtin protein impairs mitochondrial axonal transport in cultured hippocampal neurons.

    PubMed

    Tian, Jun; Yan, Ya-Ping; Zhou, Rui; Lou, Hui-Fang; Rong, Ye; Zhang, Bao-Rong

    2014-02-01

    Huntington's disease (HD) is an autosomal dominant, progressive, neurodegenerative disorder caused by an unstable expansion of CAG repeats (>35 repeats) within exon 1 of the interesting transcript 15 (IT15) gene. This gene encodes a protein called Huntingtin (Htt), and mutation of the gene results in a polyglutamine (polyQ) near the N-terminus of Htt. The N-terminal fragments of mutant Htt (mHtt), which tend to aggregate, are sufficient to cause HD. Whether these aggregates are causal or protective for HD remains hotly debated. Dysfunctional mitochondrial axonal transport is associated with HD. It remains unknown whether the soluble or aggregated form of mHtt is the primary cause of the impaired mitochondrial axonal transport in HD pathology. Here, we investigated the impact of soluble and aggregated N-terminal fragments of mHtt on mitochondrial axonal transport in cultured hippocampal neurons. We found that the N-terminal fragment of mHtt formed aggregates in almost half of the transfected neurons. Overexpression of the N-terminal fragment of mHtt decreased the velocity of mitochondrial axonal transport and mitochondrial mobility in neurons regardless of whether aggregates were formed. However, the impairment of mitochondrial axonal transport in neurons expressing the soluble and aggregated N-terminal fragments of mHtt did not differ. Our findings indicate that both the soluble and aggregated N-terminal fragments of mHtt impair mitochondrial axonal transport in cultured hippocampal neurons. We predict that dysfunction of mitochondrial axonal transport is an early-stage event in the progression of HD, even before mHtt aggregates are formed.

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

    PubMed

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

    2013-08-01

    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.

  2. A Cdk5-dependent switch regulates Lis1/Ndel1/dynein-driven organelle transport in adult axons

    PubMed Central

    Pandey, Jai P; Smith, Deanna S

    2011-01-01

    Lissencephaly is a human developmental brain abnormality caused by LIS1 haploinsufficiency. This disorder is in large part attributed to altered mitosis and migration in the developing brain. LIS1 and an interacting protein, NDEL1, bind to cytoplasmic dynein, a microtubule motor protein. While the tripartite complex is clearly important for developmental events, we are intrigued by the fact that Lis1 and Ndel1 expression remain high in the adult mouse nervous system. Dynein plays a crucial role in retrograde axonal transport, a process that is utilized by mature neurons. Here we monitored acidic organelles moving in axons of adult rat sensory neurons to determine if Lis1 and Ndel1 contribute to axonal transport. Lis1 RNAi significantly reduced axon transport of these organelles. Ndel1 RNAi had little impact, but combined Lis1 and Ndel1 RNAi caused a more severe phenotype than Lis1 RNAi alone, essentially shutting down transport. Lis1 overexpression stimulated retrograde transport, while a Lis1 dynein-binding mutant severely disrupted transport. Overexpression of Ndel1 or a Lis1 Ndel1-binding mutant only mildly perturbed transport. However, expressing a mutant Ndel1 lacking key phosphorylation sites shut down transport completely, as did a dominant negative Cdk5 construct. We propose that, in axons, unphosphorylated Ndel1 inhibits dynein’s capacity to transport acidic organelles. Phosphorylation of Ndel1 by Cdk5 not only reduces this inhibition but also allows Lis1 to further stimulate dynein’s cargo transport capacity. Our data raise the possibility that defects in a Lis1/Ndel1 regulatory switch could contribute to neurodegenerative diseases linked to axonal pathology in adults. PMID:22114287

  3. Calcium/calmodulin-dependent protein kinase IIbeta isoform is expressed in motor neurons during axon outgrowth and is part of slow axonal transport.

    PubMed

    Lund, Linda M; McQuarrie, Irvine G

    2002-03-15

    Previously, we identified calcium/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) mRNA in spinal motor neurons with 372 bp inserted in what corresponds to the "association" domain of the protein. This was interesting because known additions and deletions to CaMKIIbeta mRNA are usually less than 100 bp in size and found in the "variable" region. Changes in the association domain of CaMKIIbeta could influence substrate specificity, activity or intracellular targeting. We show that three variations of this insert are found in CNS neurons or sciatic motor neurons of Sprague-Dawley rats. We used PCR and nucleic acid sequencing to identify inserts of 114, 243, or 372 bases. We also show that addition of the 372 bases is associated with outgrowth of the axon (the standard CaMKIIbeta downregulates when axon outgrowth occurs). Radiolabeling, immunoblots, and 2D PAGE identified this larger CaMKIIbeta as part of the group of soluble proteins moving at the slowest rate of axonal transport (SCa) in sciatic motor neurons (similar1 mm/day). This group is composed mainly of structural proteins (e.g., tubulin) used to assemble the cytoskeleton of regrowing axons.

  4. The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons

    PubMed Central

    Chen, Weizhi; Mahadomrongkul, Veeravan; Berger, Urs V.; Bassan, Merav; DeSilva, Tara; Tanaka, Kohichi; Irwin, Nina; Aoki, Chiye; Rosenberg, Paul A.

    2010-01-01

    GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3′-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14–29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals. PMID:14762132

  5. Studies on Axonal Transport in an Animal Model for Gulf War Syndrome

    DTIC Science & Technology

    2008-07-01

    Biol. 12:1496–1501. Weil, D ., L. Garcon, M. Harper, D . Dumenil, F . Dautry, and M. Kress. 2002. Targeting the kinesin Eg5 to monitor siRNA...targets to the centrosome using a WD40-containing subunit. Cell 93:277–287. Hazan J, Fonknechten N, Mavel D , Paternotte C, Samson D , Artiguenave F ...Axonal transport of microtubules: the long and short of it. Traffic 7, 490–498. Buster, D ., McNally, K., and McNally, F . J. (2002). Katanin inhibition

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

    PubMed Central

    Knowlton, Wendy M.; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D.; Jin, Yishi

    2017-01-01

    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 to the cellular

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

    PubMed

    Knowlton, Wendy M; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D; Jin, Yishi

    2017-01-01

    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 to the cellular

  8. Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

    PubMed Central

    Schwab, ME; Suda, K; Thoenen, H

    1979-01-01

    The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals. PMID:92475

  9. Amyotrophic lateral sclerosis-associated mutant SOD1 inhibits anterograde axonal transport of mitochondria by reducing Miro1 levels.

    PubMed

    Moller, Annekathrin; Bauer, Claudia S; Cohen, Rebecca N; Webster, Christopher P; De Vos, Kurt J

    2017-09-14

    Defective axonal transport is an early neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously shown that ALS-associated mutations in Cu/Zn superoxide dismutase 1 (SOD1) impair axonal transport of mitochondria in motor neurons isolated from SOD1 G93A transgenic mice and in ALS mutant SOD1 transfected cortical neurons, but the underlying mechanisms remained unresolved.The outer mitochondrial membrane protein mitochondrial Rho GTPase 1 (Miro1) is a master regulator of mitochondrial axonal transport in response to cytosolic calcium (Ca2+) levels ([Ca2+]c) and mitochondrial damage. Ca2+ binding to Miro1 halts mitochondrial transport by modifying its interaction with kinesin-1 whereas mitochondrial damage induces Phosphatase and Tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and Parkin-dependent degradation of Miro1 and consequently stops transport.To identify the mechanism underlying impaired axonal transport of mitochondria in SOD1-related ALS we investigated [Ca2+]c and Miro1 levels in ALS mutant SOD1 expressing neurons. We found that expression of ALS mutant SOD1 reduced the level of endogenous Miro1 but did not affect [Ca2+]c. ALS mutant SOD1 induced reductions in Miro1 levels were Parkin dependent. Moreover, both overexpression of Miro1 and ablation of PINK1 rescued the mitochondrial axonal transport deficit in ALS mutant SOD1-expressing cortical and motor neurons.Together these results provide evidence that ALS mutant SOD1 inhibits axonal transport of mitochondria by inducing PINK1/Parkin-dependent Miro1 degradation. © The Author 2017. Published by Oxford University Press.

  10. Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth

    PubMed Central

    1987-01-01

    We have examined the movements, composition, and cellular origin of phase-dense varicosities in cultures of chick sympathetic and sensory neurons. These organelles are variable in diameter (typically between 0.2 and 2 microns) and undergo saltatory movements both towards and away from the neuronal cell body. Their mean velocities vary inversely with the size of the organelle and are greater in the retrograde than the anterograde direction. Organelles stain with the lipophilic dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine and with antibodies to cytoskeletal components. In cultures double-stained with antibodies to alpha-tubulin and 70-kD neurofilament protein (NF-L), approximately 40% of the organelles stain for tubulin, 30% stain for NF- L, 10% stain for both tubulin and NF-L, and 40% show no staining with either antibody. The association of cytoskeletal proteins with the organelles shows that these proteins are able to move by a form of rapid axonal transport. Under most culture conditions the predominant direction of movement is towards the cell body, suggesting that the organelles are produced at or near the growth cone. Retrograde movements continue in culture medium lacking protein or high molecular mass components and increase under conditions in which the advance of the growth cone is arrested. There is a fourfold increase in the number of organelles moving retrogradely in neurites that encounter a substratum-associated barrier to elongation; retrograde movements increase similarly in cultures exposed to cytochalasin at levels known to block growth cone advance. No previously described organelle shows behavior coordinated with axonal growth in this way. We propose that the organelles contain membrane and cytoskeletal components that have been delivered to the growth cone, by slow or fast anterograde transport, in excess of the amounts required to synthesize more axon. In view of their rapid mobility and variable contents, we suggest that they

  11. Quantitative analysis of APP axonal transport in neurons: role of JIP1 in enhanced APP anterograde transport

    PubMed Central

    Chiba, Kyoko; Araseki, Masahiko; Nozawa, Keisuke; Furukori, Keiko; Araki, Yoichi; Matsushima, Takahide; Nakaya, Tadashi; Hata, Saori; Saito, Yuhki; Uchida, Seiichi; Okada, Yasushi; Nairn, Angus C.; Davis, Roger J.; Yamamoto, Tohru; Kinjo, Masataka; Taru, Hidenori; Suzuki, Toshiharu

    2014-01-01

    Alzheimer's β-amyloid precursor protein (APP) associates with kinesin-1 via JNK-interacting protein 1 (JIP1); however, the role of JIP1 in APP transport by kinesin-1 in neurons remains unclear. We performed a quantitative analysis to understand the role of JIP1 in APP axonal transport. In JIP1-deficient neurons, we find that both the fast velocity (∼2.7 μm/s) and high frequency (66%) of anterograde transport of APP cargo are impaired to a reduced velocity (∼1.83 μm/s) and a lower frequency (45%). We identified two novel elements linked to JIP1 function, located in the central region of JIP1b, that interact with the coiled-coil domain of kinesin light chain 1 (KLC1), in addition to the conventional interaction of the JIP1b 11–amino acid C-terminal (C11) region with the tetratricopeptide repeat of KLC1. High frequency of APP anterograde transport is dependent on one of the novel elements in JIP1b. Fast velocity of APP cargo transport requires the C11 domain, which is regulated by the second novel region of JIP1b. Furthermore, efficient APP axonal transport is not influenced by phosphorylation of APP at Thr-668, a site known to be phosphorylated by JNK. Our quantitative analysis indicates that enhanced fast-velocity and efficient high-frequency APP anterograde transport observed in neurons are mediated by novel roles of JIP1b. PMID:25165140

  12. Herpes simplex virus gE/gI extracellular domains promote axonal transport and spread from neurons to epithelial cells.

    PubMed

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

    2014-10-01

    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

  13. Transfer of axonally transported phospholipids into myelin isolated from the rabbit optic pathway

    SciTech Connect

    Alberghina, M.; Viola, M.; Giuffrida, A.M.

    1982-02-01

    The contribution of the axonal transport to the biosynthesis of myelin phospholipids was investigated in the rabbit optic pathway. A double labeling technique was used. The same animals were injected with one isotope intravitreally and the other intraventricularly. This procedure allows double labeling of the optic nerves, optic tracts, lateral geniculate bodies (LGB), and superior colliculus (SC). The precursors simultaneously injected were: (1-/sup 14/C)palmitate (15 microCi intravitreally in both eyes or 50 microCi intraventricularly) and (2-/sup 3/H)glycerol (50 microCi intravitreally in both eyes of 100 microCi intraventricularly). Twenty four hours and 10 days after the injections, myelin was purified from pooled optic nerves and optic tracts as well as from pooled LGBs or SCs. The phospholipids were extracted and then separated by thin-layer chromatography; the specific radioactivity of the various classes of phospholipids was determined. Using both administration routes of C- or /sup 3/H-precursors, the distribution of label and specific radioactivity of myelin phospholipids in the retina and in all other optic structures were very similar. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphoinositol were preferentially labeled with both precursors. These results suggest that, in the rabbit optic pathway the phospholipids synthesized in the retinal ganglion cells and transported along the axons, could undergo transaxonal transfer into myelin.

  14. Retrograde axonal transport of /sup 125/I-nerve growth factor in rat ileal mesenteric nerves. Effect of streptozocin diabetes

    SciTech Connect

    Schmidt, R.E.; Plurad, S.B.; Saffitz, J.E.; Grabau, G.G.; Yip, H.K.

    1985-12-01

    The retrograde axonal transport of intravenously (i.v.) administered /sup 125/I-nerve growth factor (/sup 125/I-NGF) was examined in mesenteric nerves innervating the small bowel of rats with streptozocin (STZ) diabetes using methods described in detail in the companion article. The accumulation of /sup 125/I-NGF distal to a ligature on the ileal mesenteric nerves of diabetic animals was 30-40% less than in control animals. The inhibition of accumulation of /sup 125/I-NGF in diabetic animals was greater at a ligature tied 2 h after i.v. administration than at a ligature tied after 14 h, which suggests that the diabetic animals may have a lag in initiation of NGF transport in the terminal axon or retardation of transport at some site along the axon. The /sup 125/I-NGF transport defect was observed as early as 3 days after the induction of diabetes, a time before the development of structural axonal lesions, and did not worsen at later times when dystrophic axonopathy is present. Both the ileal mesenteric nerves, which eventually develop dystrophic axonopathy in experimental diabetes, and the jejunal mesenteric nerves, which never develop comparable structural alterations, showed similar /sup 125/I-NGF transport deficits, suggesting that the existence of the transport abnormality does not predict the eventual development of dystrophic axonal lesions. Autoradiographic localization of /sup 125/I-NGF in the ileal mesenteric nerves of animals that had been diabetic for 11-13 mo demonstrated decreased amounts of /sup 125/I-NGF in transit in unligated paravascular nerve fascicles. There was, however, no evidence for focal retardation of transported /sup 125/I-NGF at the sites of dystrophic axonal lesions.

  15. Localization of axonally transported 125I-wheat germ agglutinin beneath the plasma membrane of chick retinal ganglion cells

    PubMed Central

    1983-01-01

    The distribution of 125I-wheat germ agglutinin (WGA) transported by axons of chick retinal ganglion cells to layer d of the optic tectum was studied by electron microscopic autoradiography. We found that 52% of the radioactivity was located in axons and axon terminals in the contralateral optic tectum 22 h after intravitreal injection of affinity-purified 125I-WGA. Axons comprised 43% of the volume of layer d. Dendrites, glial cells, and neuron cell bodies contained 20%, 17%, and 3% of the label, whereas these structures comprised 24%, 21%, and 2% of the tissue volume, respectively. We also measured the distances between the autoradiographic silver grains and the plasma membranes of these profiles, and compared observed distributions of grains to theoretical distributions computed for band-shaped sources at various distances from the plasma membranes. This analysis revealed that the radioactive source within axons was distributed in a band of cytoplasm extending in from the plasma membrane a distance of 63 nm. Because WGA is known to bind to specific membrane glycoconjugates, we infer that at least some glycoconjugates may be concentrated within an annular region of cytoplasm just beneath the axonal plasma membrane after axoplasmic transport from the neuron cell body. PMID:6187749

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

    PubMed

    Segal, Michal; Soifer, Ilya; Petzold, Heike; Howard, Jonathon; Elbaum, Michael; Reiner, Orly

    2012-03-15

    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.

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

    PubMed Central

    Segal, Michal; Soifer, Ilya; Petzold, Heike; Howard, Jonathon; Elbaum, Michael; Reiner, Orly

    2012-01-01

    Summary 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. PMID:23213412

  18. Quantitative measurements and modeling of cargo–motor interactions during fast transport in the living axon

    PubMed Central

    Seamster, Pamela E; Loewenberg, Michael; Pascal, Jennifer; Chauviere, Arnaud; Gonzales, Aaron; Cristini, Vittorio; Bearer, Elaine L

    2013-01-01

    The kinesins have long been known to drive microtubule-based transport of sub-cellular components, yet the mechanisms of their attachment to cargo remain a mystery. Several different cargo-receptors have been proposed based on their in vitro binding affinities to kinesin-1. Only two of these—phosphatidyl inositol, a negatively charged lipid, and the carboxyl terminus of the amyloid precursor protein (APP-C), a trans-membrane protein—have been reported to mediate motility in living systems. A major question is how these many different cargo, receptors and motors interact to produce the complex choreography of vesicular transport within living cells. Here we describe an experimental assay that identifies cargo–motor receptors by their ability to recruit active motors and drive transport of exogenous cargo towards the synapse in living axons. Cargo is engineered by derivatizing the surface of polystyrene fluorescent nanospheres (100 nm diameter) with charged residues or with synthetic peptides derived from candidate motor receptor proteins, all designed to display a terminal COOH group. After injection into the squid giant axon, particle movements are imaged by laser-scanning confocal time-lapse microscopy. In this report we compare the motility of negatively charged beads with APP-C beads in the presence of glycine-conjugated non-motile beads using new strategies to measure bead movements. The ensuing quantitative analysis of time-lapse digital sequences reveals detailed information about bead movements: instantaneous and maximum velocities, run lengths, pause frequencies and pause durations. These measurements provide parameters for a mathematical model that predicts the spatiotemporal evolution of distribution of the two different types of bead cargo in the axon. The results reveal that negatively charged beads differ from APP-C beads in velocity and dispersion, and predict that at long time points APP-C will achieve greater progress towards the presynaptic

  19. Quantitative measurements and modeling of cargo-motor interactions during fast transport in the living axon

    NASA Astrophysics Data System (ADS)

    Seamster, Pamela E.; Loewenberg, Michael; Pascal, Jennifer; Chauviere, Arnaud; Gonzales, Aaron; Cristini, Vittorio; Bearer, Elaine L.

    2012-10-01

    The kinesins have long been known to drive microtubule-based transport of sub-cellular components, yet the mechanisms of their attachment to cargo remain a mystery. Several different cargo-receptors have been proposed based on their in vitro binding affinities to kinesin-1. Only two of these—phosphatidyl inositol, a negatively charged lipid, and the carboxyl terminus of the amyloid precursor protein (APP-C), a trans-membrane protein—have been reported to mediate motility in living systems. A major question is how these many different cargo, receptors and motors interact to produce the complex choreography of vesicular transport within living cells. Here we describe an experimental assay that identifies cargo-motor receptors by their ability to recruit active motors and drive transport of exogenous cargo towards the synapse in living axons. Cargo is engineered by derivatizing the surface of polystyrene fluorescent nanospheres (100 nm diameter) with charged residues or with synthetic peptides derived from candidate motor receptor proteins, all designed to display a terminal COOH group. After injection into the squid giant axon, particle movements are imaged by laser-scanning confocal time-lapse microscopy. In this report we compare the motility of negatively charged beads with APP-C beads in the presence of glycine-conjugated non-motile beads using new strategies to measure bead movements. The ensuing quantitative analysis of time-lapse digital sequences reveals detailed information about bead movements: instantaneous and maximum velocities, run lengths, pause frequencies and pause durations. These measurements provide parameters for a mathematical model that predicts the spatiotemporal evolution of distribution of the two different types of bead cargo in the axon. The results reveal that negatively charged beads differ from APP-C beads in velocity and dispersion, and predict that at long time points APP-C will achieve greater progress towards the presynaptic

  20. Quantitative measurements and modeling of cargo-motor interactions during fast transport in the living axon.

    PubMed

    Seamster, Pamela E; Loewenberg, Michael; Pascal, Jennifer; Chauviere, Arnaud; Gonzales, Aaron; Cristini, Vittorio; Bearer, Elaine L

    2012-10-01

    The kinesins have long been known to drive microtubule-based transport of sub-cellular components, yet the mechanisms of their attachment to cargo remain a mystery. Several different cargo-receptors have been proposed based on their in vitro binding affinities to kinesin-1. Only two of these-phosphatidyl inositol, a negatively charged lipid, and the carboxyl terminus of the amyloid precursor protein (APP-C), a trans-membrane protein-have been reported to mediate motility in living systems. A major question is how these many different cargo, receptors and motors interact to produce the complex choreography of vesicular transport within living cells. Here we describe an experimental assay that identifies cargo-motor receptors by their ability to recruit active motors and drive transport of exogenous cargo towards the synapse in living axons. Cargo is engineered by derivatizing the surface of polystyrene fluorescent nanospheres (100 nm diameter) with charged residues or with synthetic peptides derived from candidate motor receptor proteins, all designed to display a terminal COOH group. After injection into the squid giant axon, particle movements are imaged by laser-scanning confocal time-lapse microscopy. In this report we compare the motility of negatively charged beads with APP-C beads in the presence of glycine-conjugated non-motile beads using new strategies to measure bead movements. The ensuing quantitative analysis of time-lapse digital sequences reveals detailed information about bead movements: instantaneous and maximum velocities, run lengths, pause frequencies and pause durations. These measurements provide parameters for a mathematical model that predicts the spatiotemporal evolution of distribution of the two different types of bead cargo in the axon. The results reveal that negatively charged beads differ from APP-C beads in velocity and dispersion, and predict that at long time points APP-C will achieve greater progress towards the presynaptic

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

    PubMed

    Magen, Iddo; Gozes, Illana

    2013-12-01

    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.

  2. The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system.

    PubMed

    Gindhart, Joseph G; Chen, Jinyun; Faulkner, Melissa; Gandhi, Rita; Doerner, Karl; Wisniewski, Tiffany; Nandlestadt, Aline

    2003-08-01

    Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.

  3. Retrograde axonal transport of herpes simplex virus: Evidence for a single mechanism and a role for tegument

    PubMed Central

    Bearer, E. L.; Breakefield, X. O.; Schuback, D.; Reese, T. S.; LaVail, J. H.

    2000-01-01

    Herpes simplex virus type I (HSV) typically enters peripheral nerve terminals and then travels back along the nerve to reach the neuronal cell body, where it replicates or enters latency. To monitor axoplasmic transport of HSV, we used the giant axon of the squid, Loligo pealei, a well known system for the study of axoplasmic transport. To deliver HSV into the axoplasm, viral particles stripped of their envelopes by detergent were injected into the giant axon, thereby bypassing the infective process. Labeling the viral tegument protein, VP16, with green fluorescent protein allowed viral particles moving inside the axon to be imaged by confocal microscopy. Viral particles moved 2.2 ± 0.26 μm/sec in the retrograde direction, a rate comparable to that of the transport of endogenous organelles and of virus in mammalian neurons in culture. Electron microscopy confirmed that 96% of motile (stripped) viral particles had lost their envelope but retained tegument, and Western blot analysis revealed that these particles had retained protein from capsid but not envelope. We conclude that (i) HSV recruits the squid retrograde transport machinery; (ii) viral tegument and capsid but not envelope are sufficient for this recruitment; and (iii) the giant axon of the squid provides a unique system to dissect the viral components required for transport and to identify the cellular transport mechanisms they recruit. PMID:10884436

  4. Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo

    PubMed Central

    White, Joseph A.; Anderson, Eric; Zimmerman, Katherine; Zheng, Kan Hong; Rouhani, Roza; Gunawardena, Shermali

    2015-01-01

    Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. Here we analyze the in vivo movement of 16 Rab GTPases within Drosophila larval axons and show that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, our findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for our understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease. PMID:26450517

  5. Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo.

    PubMed

    White, Joseph A; Anderson, Eric; Zimmerman, Katherine; Zheng, Kan Hong; Rouhani, Roza; Gunawardena, Shermali

    2015-12-20

    Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. Here we analyze the in vivo movement of 16 Rab GTPases within Drosophila larval axons and show that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, our findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for our understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. A Single-File Model for Potassium Transport in Squid Giant Axon

    PubMed Central

    Kohler, H. -H.

    1977-01-01

    A physical model for potassium transport in squid giant axon is proposed. The model is designed to explain the empirical data given by the Hodgkin-Huxley model and related experiments. It is assumed that K+ moves across the axon membrane by single-file diffusion through narrow pores. In the model a pore has three negatively charged sites that can be occupied alternatively by K+ or by a gating particle, GP++, coming from the external surface. GP++ is considered to be part of the membrane rather than a diffusible component of the surrounding solutions. A high activation barrier for GP++ is supposed at the inner membrane border so that it cannot change over to the internal surface. Therefore potassium diffusion can be blocked by GP++ penetrating into the pores. This mechanism controls the dynamic behaviour of the model. The time-dependent probabilities of the pore states are described by a system of differential equations. The rate constants in these equations depend on the ionic concentrations, the membrane voltage, and the electrostatic interaction between ions in a single pore. Detailed computational tests for normal composition of external and internal solutions show that the model agrees remarkably well with the stationary and dynamic behaviour of the Hodgkin-Huxley model. However, the hyperpolarization delay is not reproduced. A structural modification, concerning this delay and the way in which GP++ is attached to the membrane, is proposed, and the qualitative behavior of the model at varied external and internal concentrations is discussed. PMID:880331

  7. Soluble Conformers of Aβ and Tau Alter Selective Proteins Governing Axonal Transport

    PubMed Central

    Sherman, Mathew A.; LaCroix, Michael; Amar, Fatou; Larson, Megan E.; Forster, Colleen; Aguzzi, Adriano; Bennett, David A.; Ramsden, Martin

    2016-01-01

    Despite the demonstration that amyloid-β (Aβ) can trigger increased tau phosphorylation and neurofibrillary tangle (NFT) formation in vivo, the molecular link associating Aβ and tau pathologies remains ill defined. Here, we observed that exposure of cultured primary neurons to Aβ trimers isolated from brain tissue of subjects with Alzheimer's disease led to a specific conformational change of tau detected by the antibody Alz50. A similar association was supported by postmortem human brain analyses. To study the role of Aβ trimers in vivo, we created a novel bigenic Tg-Aβ+Tau mouse line by crossing Tg2576 (Tg-Aβ) and rTg4510 (Tg-Tau) mice. Before neurodegeneration and amyloidosis, apparent Aβ trimers were increased by ∼2-fold in 3-month-old Tg-Aβ and Tg-Aβ+Tau mice compared with younger mice, whereas soluble monomeric Aβ levels were unchanged. Under these conditions, the expression of soluble Alz50-tau conformers rose by ∼2.2-fold in the forebrains of Tg-Aβ+Tau mice compared with nontransgenic littermates. In parallel, APP accumulated intracellularly, suggestive of a putative dysfunction of anterograde axonal transport. We found that the protein abundance of the kinesin-1 light chain (KLC1) was reduced selectively in vivo and in vitro when soluble Aβ trimers/Alz50-tau were present. Importantly, the reduction in KLC1 was prevented by the intraneuronal delivery of Alz50 antibodies. Collectively, our findings reveal that specific soluble conformers of Aβ and tau cooperatively disrupt axonal transport independently from plaques and tangles. Finally, these results suggest that not all endogenous Aβ oligomers trigger the same deleterious changes and that the role of each assembly should be considered separately. SIGNIFICANCE STATEMENT The mechanistic link between amyloid-β (Aβ) and tau, the two major proteins composing the neuropathological lesions detected in brain tissue of Alzheimer's disease subjects, remains unclear. Here, we report that the

  8. Soluble Conformers of Aβ and Tau Alter Selective Proteins Governing Axonal Transport.

    PubMed

    Sherman, Mathew A; LaCroix, Michael; Amar, Fatou; Larson, Megan E; Forster, Colleen; Aguzzi, Adriano; Bennett, David A; Ramsden, Martin; Lesné, Sylvain E

    2016-09-14

    Despite the demonstration that amyloid-β (Aβ) can trigger increased tau phosphorylation and neurofibrillary tangle (NFT) formation in vivo, the molecular link associating Aβ and tau pathologies remains ill defined. Here, we observed that exposure of cultured primary neurons to Aβ trimers isolated from brain tissue of subjects with Alzheimer's disease led to a specific conformational change of tau detected by the antibody Alz50. A similar association was supported by postmortem human brain analyses. To study the role of Aβ trimers in vivo, we created a novel bigenic Tg-Aβ+Tau mouse line by crossing Tg2576 (Tg-Aβ) and rTg4510 (Tg-Tau) mice. Before neurodegeneration and amyloidosis, apparent Aβ trimers were increased by ∼2-fold in 3-month-old Tg-Aβ and Tg-Aβ+Tau mice compared with younger mice, whereas soluble monomeric Aβ levels were unchanged. Under these conditions, the expression of soluble Alz50-tau conformers rose by ∼2.2-fold in the forebrains of Tg-Aβ+Tau mice compared with nontransgenic littermates. In parallel, APP accumulated intracellularly, suggestive of a putative dysfunction of anterograde axonal transport. We found that the protein abundance of the kinesin-1 light chain (KLC1) was reduced selectively in vivo and in vitro when soluble Aβ trimers/Alz50-tau were present. Importantly, the reduction in KLC1 was prevented by the intraneuronal delivery of Alz50 antibodies. Collectively, our findings reveal that specific soluble conformers of Aβ and tau cooperatively disrupt axonal transport independently from plaques and tangles. Finally, these results suggest that not all endogenous Aβ oligomers trigger the same deleterious changes and that the role of each assembly should be considered separately. The mechanistic link between amyloid-β (Aβ) and tau, the two major proteins composing the neuropathological lesions detected in brain tissue of Alzheimer's disease subjects, remains unclear. Here, we report that the trimeric Aβ species induce

  9. Transport characteristics of urea transporter-B.

    PubMed

    Yang, Baoxue

    2014-01-01

    UT-B represents the major urea transporter in erythrocytes, in addition to being expressed in kidney descending vasa recta, brain, spleen, ureter, bladder, and testis. Expression of urea transporter UT-B confers high urea permeability to mammalian erythrocytes. Erythrocyte membranes are also permeable to various urea analogues, suggesting common transport pathways for urea and structurally similar solutes. UT-B is highly permeable to urea and the chemical analogues formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, each with a Ps > 5.0 × 10(-6) cm/s at 10 °C. The amides formamide, acetamide, acrylamide, and butyramide efficiently diffuse across lipid bilayers. The urea analogues dimethylurea, acryalmide, methylurea, thiourea, and methylformamide inhibit UT-B-mediated urea transport by >60 % by a pore-blocking mechanism. UT-B is also a water channel in erythrocytes and has a single-channel water permeability that is similar to aquaporin-1. Whether UT-B is an NH3 channel still needs further study. Urea permeability (Purea) in erythrocytes differs between different mammals. Carnivores (dog, fox, cat) exhibit high Purea. In contrast, herbivores (cow, donkey, sheep) show much lower Purea. Erythrocyte Purea in human and pig (omnivores) was intermediate. Rodents and lagomorphs (mouse, rat, rabbit) have Purea intermediate between carnivores and omnivores. Birds that do not excrete urea and do not express UT-B in their erythrocytes have very low values. In contrast to Purea, water permeability is relatively similar in all mammals studied. This chapter will provide information about the transporter characteristics of UT-B.

  10. Failure of unilateral carotid artery ligation to affect pressure-induced interruption of rapid axonal transport in primate optic nerves.

    PubMed

    Radius, R L; Schwartz, E L; Anderson, D R

    1980-02-01

    Previous experiments showed that optic nerve axonal transport can be blocked at the level of the lamina cribrosa by elevated intraocular pressure. In an effort to discover if this blockage might be secondary to pressure-induced ischemia, we studied the effect of unilateral common carotid artery ligation upont the pressure-induced interruption of axonal transport. In 13 owl monkeys (Aotus trivirgatus), the right common carotid artery was ligated within the anterior cervical triangle. Three days later, ophtalmodynomometry was performed on all experimental eyes. In nine of the 13 animals, this estimate of ophthalmic artery pressure was 10 to 20 mm Hg less in the right compared to the left eye. Optic nerve axonal transport was studied in right and left eyes during 5 hours of increased intraocular pressure (ocular pressure 35 mm Hg less than mean femoral artery blood pressure). No significant difference in the extent to which the transport mechanisms were interrupted could be demonstrated when comparing right and left eyes of the experimental animals. These observations fail to support a vascular mechanism for this pressure-induced interruption of axonal transport.

  11. Dynactin regulates bidirectional transport of dense-core vesicles in the axon and dendrites of cultured hippocampal neurons.

    PubMed

    Kwinter, D M; Lo, K; Mafi, P; Silverman, M A

    2009-09-15

    A critical aspect of nerve cell function is peptidergic secretion involving the packaging, transport, and processing of a large group of peptide hormones and other signaling molecules, e.g. brain-derived neurotrophic factor (BDNF). Dense-core vesicles (DCVs) are the organelles that transport these molecules to release sites in both the axon and dendrites of pyramidal neurons. DCVs exhibit complex transport behavior, where these organelles move bidirectionally, reverse direction, pause intermittently, and vary in velocities and run lengths. A key objective in the field of organelle transport is to define the molecules that mediate transport. This study investigated the role of dynactin, a putative opposite-polarity motor coordinator, in the microtubule-based transport of DCVs in primary cultured hippocampal neurons. First, by live cell imaging, we showed similar microtubule-based transport of BDNF, neuropeptide Y (NPY), and tissue plasminogen activator (tPA), consistent with the co-packaging of these DCV cargoes. However, we found higher DCV velocities in both the axon and dendrites than those of previous neuronal studies likely due to faster image acquisition times. Then, using well-characterized dynactin disruptors we demonstrate the need for dynactin in bidirectional transport where overexpression of both p50/dynamitin and the first coiled-coil domain of p150(Glued) (CC1) reduces the flux of DCVs in both directions in the axon and dendrites. We also observed that only CC1 reduces axonal and dendritic run lengths. These results suggest different functions for p50 and p150 in the dynactin complex in DCV transport. These findings are significant because they demonstrate that dynactin functions as a motor coordinator for the transport of DCVs in primary cultured rat hippocampal neurons.

  12. Morphological evidence for a transport of ribosomes from Schwann cells to regenerating axons.

    PubMed

    Court, Felipe A; Midha, Rajiv; Cisterna, Bruno A; Grochmal, Joey; Shakhbazau, Antos; Hendriks, William T; Van Minnen, Jan

    2011-10-01

    Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector-mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.

  13. Pressure-induced fast axonal transport abnormalities and the anatomy at the lamina cribrosa in primate eyes.

    PubMed

    Radius, R L

    1983-03-01

    In ten owl monkey eyes (Aotus trivirgatus) the location of pressure-induced (perfusion pressure 35 mmHg) axonal transport abnormalities was determined by the examination of serial step cross-section tissue radio autographs from the optic nerve head. The degree of the local transport interruption did not correlate with the fiber bundle cross-section area, the shape of the laminar pores or the density of the inter-bundle septa in that region.

  14. Modeling Huntington disease in Drosophila: Insights into axonal transport defects and modifiers of toxicity.

    PubMed

    Krench, Megan; Littleton, J Troy

    2013-01-01

    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.

  15. The use of whole-mount preparations of nerves labelled with axonally transported radioactive proteins to study regeneration.

    PubMed

    Edström, A; Sjöberg, J; Kanje, M

    1986-03-01

    A method for studying the process of regeneration in the frog sciatic nerve is presented. The outgrowth of sensory axons was determined by assaying the distribution of axonally transported radioactive proteins by fluorography of whole-mount nerve preparations. After labelling, the nerves were fixed at their in vivo lengths, impregnated with a fluorographic reagent and whole-mounted on a glass-slide before exposure to an X-ray film. In this way the rate of axon elongation after nerve crush lesions could be accurately determined. Nerve regeneration preceded by a conditioning lesion will be described. After the outgrowth has been measured the method enables subsequent analysis of, for instance, the protein composition in selected parts of the same nerve. The technique should be broadly applicable for studying nerve regeneration.

  16. Critical role of JSAP1 and JLP in axonal transport in the cerebellar Purkinje cells of mice.

    PubMed

    Sato, Tokiharu; Ishikawa, Momoe; Yoshihara, Toru; Nakazato, Ryota; Higashida, Haruhiro; Asano, Masahide; Yoshioka, Katsuji

    2015-09-14

    JNK/stress-activated protein kinase-associated protein 1 (JSAP1) and JNK-associated leucine zipper protein (JLP) are structurally related scaffolding proteins that are highly expressed in the brain. Here, we found that JSAP1 and JLP play functionally redundant and essential roles in mouse cerebellar Purkinje cell (PC) survival. Mice containing PCs with deletions in both JSAP1 and JLP exhibited PC axonal dystrophy, followed by gradual, progressive neuronal loss. Kinesin-1 cargoes accumulated selectively in the swollen axons of Jsap1/Jlp-deficient PCs. In addition, autophagy inactivation in these mice markedly accelerated PC degeneration. These findings suggest that JSAP1 and JLP play critical roles in kinesin-1-dependent axonal transport, which prevents brain neuronal degeneration. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Deficits in axonal transport in hippocampal-based circuitry and the visual pathway in APP knock-out animals witnessed by manganese enhanced MRI

    PubMed Central

    Gallagher, Joseph J.; Zhang, Xiaowei; Ziomek, Greg; Jacobs, Russell E.; Bearer, Elaine L.

    2012-01-01

    Mounting evidence implicates axonal transport defects, typified by the presence of axonal varicosities with aberrant accumulations of cargo, as an early event in Alzheimer’s disease (AD) pathogenesis. Work identifying amyloid precursor protein (APP) as a vesicular motor receptor for anterograde axonal transport further implicates axonal transport in AD. Manganese-enhanced MRI (MEMRI) detects axonal transport dynamics in preclinical studies. Here we pursue an understanding of the role of APP in axonal transport in the central nervous system by applying MEMRI to hippocampal circuitry and to the visual pathway in living mice homozygous for either wild type or a deletion in the APP gene (n = 12 for each genotype). Following intra-ocular or stereotaxic hippocampal injection, we performed time-lapse MRI to detect Mn2+ transport. Three dimensional whole brain datasets were compared on a voxel-wise basis using within-group pair-wise analysis. Quantification of transport to structures connected to injection sites via axonal fiber tracts was also performed. Histology confirmed consistent placement of hippocampal injections and no observable difference in glial-response to the injections. APP −/− mice had significantly reduced transport from the hippocampus to the septal nuclei and amygdala after 7 hours and reduced transport to the contralateral hippocampus after 25 hours; axonal transport deficits in the APP −/− animals were also identified in the visual pathway. These data support a system-wide role for APP in axonal transport within the central nervous system and demonstrate the power of MEMRI for assessing neuronal circuitry involved in memory and learning. PMID:22500926

  18. Deficits in axonal transport in hippocampal-based circuitry and the visual pathway in APP knock-out animals witnessed by manganese enhanced MRI.

    PubMed

    Gallagher, Joseph J; Zhang, Xiaowei; Ziomek, Gregory J; Jacobs, Russell E; Bearer, Elaine L

    2012-04-15

    Mounting evidence implicates axonal transport defects, typified by the presence of axonal varicosities with aberrant accumulations of cargo, as an early event in Alzheimer's disease (AD) pathogenesis. Work identifying amyloid precursor protein (APP) as a vesicular motor receptor for anterograde axonal transport further implicates axonal transport in AD. Manganese-enhanced MRI (MEMRI) detects axonal transport dynamics in preclinical studies. Here we pursue an understanding of the role of APP in axonal transport in the central nervous system by applying MEMRI to hippocampal circuitry and to the visual pathway in living mice homozygous for either wild type or a deletion in the APP gene (n=12 for each genotype). Following intra-ocular or stereotaxic hippocampal injection, we performed time-lapse MRI to detect Mn(2+) transport. Three dimensional whole brain datasets were compared on a voxel-wise basis using within-group pair-wise analysis. Quantification of transport to structures connected to injection sites via axonal fiber tracts was also performed. Histology confirmed consistent placement of hippocampal injections and no observable difference in glial-response to the injections. APP-/- mice had significantly reduced transport from the hippocampus to the septal nuclei and amygdala after 7h and reduced transport to the contralateral hippocampus after 25 h; axonal transport deficits in the APP-/- animals were also identified in the visual pathway. These data support a system-wide role for APP in axonal transport within the central nervous system and demonstrate the power of MEMRI for assessing neuronal circuitry involved in memory and learning.

  19. Sensory neuropathy in progressive motor neuronopathy (pmn) mice is associated with defects in microtubule polymerization and axonal transport.

    PubMed

    Schäfer, Michael K; Bellouze, Sarah; Jacquier, Arnaud; Schaller, Sébastien; Richard, Laurence; Mathis, Stéphane; Vallat, Jean-Michel; Haase, Georg

    2016-08-04

    Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) are now recognized as multi-system disorders also involving various non-motor neuronal cell types. The precise extent and mechanistic basis of non-motor neuron damage in human ALS and ALS animal models remain however unclear. To address this, we here studied progressive motor neuronopathy (pmn) mice carrying a missense loss-of-function mutation in tubulin binding cofactor E (TBCE). These mice manifest a particularly aggressive form of motor axon dying back and display a microtubule loss, similar to that induced by human ALS-linked TUBA4A mutations. Using whole nerve confocal imaging of pmn × thy1.2-YFP16 fluorescent reporter mice and electron microscopy, we demonstrate axonal discontinuities, bead-like spheroids and ovoids in pmn suralis nerves indicating prominent sensory neuropathy. The axonal alterations qualitatively resemble those in phrenic motor nerves but do not culminate in the loss of myelinated fibers. We further show that the pmn mutation decreases the level of TBCE, impedes microtubule polymerization in dorsal root ganglion (DRG) neurons and causes progressive loss of microtubules in large and small caliber suralis axons. Live imaging of axonal transport using GFP-tagged tetanus toxin C-fragment (GFP-TTC) demonstrates defects in microtubule-based transport in pmn DRG neurons, providing a potential explanation for the axonal alterations in sensory nerves. This study unravels sensory neuropathy as a pathological feature of mouse pmn, and discusses the potential contribution of cytoskeletal defects to sensory neuropathy in human motor neuron disease.

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

    PubMed Central

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

    2014-01-01

    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

  1. Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3

    PubMed Central

    Tateno, Minako; Kato, Shinsuke; Sakurai, Takashi; Nukina, Nobuyuki; Takahashi, Ryosuke; Araki, Toshiyuki

    2009-01-01

    Mutations in the superoxide dismutase 1 (sod1) gene cause familial amyotrophic lateral sclerosis (FALS), likely due to the toxic properties of misfolded mutant SOD1 protein. Here we demonstrated that, starting from the pre-onset stage of FALS, misfolded SOD1 species associates specifically with kinesin-associated protein 3 (KAP3) in the ventral white matter of SOD1G93A-transgenic mouse spinal cord. KAP3 is a kinesin-2 subunit responsible for binding to cargos including choline acetyltransferase (ChAT). Motor axons in SOD1G93A-Tg mice also showed a reduction in ChAT transport from the pre-onset stage. By employing a novel FALS modeling system using NG108-15 cells, we showed that microtubule-dependent release of acetylcholine was significantly impaired by misfolded SOD1 species. Furthermore, such impairment was able to be normalized by KAP3 overexpression. KAP3 was incorporated into SOD1 aggregates in human FALS cases as well. These results suggest that KAP3 sequestration by misfolded SOD1 species and the resultant inhibition of ChAT transport play a role in the dysfunction of ALS. PMID:19088126

  2. Neuronal injury increases retrograde axonal transport of the neurotrophins to spinal sensory neurons and motor neurons via multiple receptor mechanisms.

    PubMed

    Curtis, R; Tonra, J R; Stark, J L; Adryan, K M; Park, J S; Cliffer, K D; Lindsay, R M; DiStefano, P S

    1998-10-01

    We investigated the retrograde axonal transport of 125I-labeled neurotrophins (NGF, BDNF, NT-3, and NT-4) from the sciatic nerve to dorsal root ganglion (DRG) sensory neurons and spinal motor neurons in normal rats or after neuronal injury. DRG neurons showed increased transport of all neurotrophins following crush injury to the sciatic nerve. This was maximal 1 day after sciatic nerve crush and returned to control levels after 7 days. 125I-BDNF transport from sciatic nerve was elevated with injection either proximal to the lesion or directly into the crush site and after transection of the dorsal roots. All neurotrophin transport was receptor-mediated and consistent with neurotrophin binding to the low-affinity neurotrophin receptor (LNR) or Trk receptors. However, transport of 125I-labeled wheat germ agglutinin also increased 1 day after sciatic nerve crush, showing that increased uptake and transport is a generalized response to injury in DRG sensory neurons. Spinal cord motor neurons also showed increased neurotrophin transport following sciatic nerve injury, although this was maximal after 3 days. The transport of 125I-NGF depended on the expression of LNR by injured motor neurons, as demonstrated by competition experiments with unlabeled neurotrophins. The absence of TrkA in normal motor neurons or after axotomy was confirmed by immunostaining and in situ hybridization. Thus, increased transport of neurotrophic factors after neuronal injury is due to multiple receptor-mediated mechanisms including general increases in axonal transport capacity. Copyright 1998 Academic Press.

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

    SciTech Connect

    Maier, C.E.

    1989-01-01

    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.

  4. Axonal transport studied in a single vertebrate neuron: the giant electromotor neuron of the electric catfish, Malapterurus electricus.

    PubMed

    Zimmermann, H; Tashiro, T; Komiya, Y; Kurokawa, M

    1989-02-01

    Axonal transport was studied using a single vertebrate neuron, the giant electromotor neuron of the electric catfish, Malapterurus electricus. The electric organs of this strongly electric fish are innervated by two neurons whose axons form one electric nerve each. After injection of [35S]methionine into the spinal cord at the level of the two perikarya radioactively labelled material is exported by fast flow as a small wave with a velocity of 5.8 mm/h and a somal release time of 91 min (29 degrees C). Slow flow investigated between 15 and 39 days had a velocity of 1.36 mm/d at 29 degrees C. Analysis of radiolabelled proteins by polyacrylamide gel electrophoresis revealed different patterns of labelling between slow and fast flow. The relative molecular mass of the two major proteins labelled on slow flow correspond to actin and tubulin. Labelled proteins of higher relative molecular mass may correspond to neurofilament proteins. Our results suggest that this vertebrate single-neuron and single-axon system can be used successfully for axonal transport studies.

  5. Limited Trafficking of a Neurotropic Virus Through Inefficient Retrograde Axonal Transport and the Type I Interferon Response

    PubMed Central

    Lancaster, Karen Z.; Pfeiffer, Julie K.

    2010-01-01

    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) α/β 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 α/β 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. PMID:20221252

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

    PubMed

    Lancaster, Karen Z; Pfeiffer, Julie K

    2010-03-05

    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.

  7. Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

    NASA Technical Reports Server (NTRS)

    Tsai, M. Y.; Morfini, G.; Szebenyi, G.; Brady, S. T.

    2000-01-01

    The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

  8. Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

    NASA Technical Reports Server (NTRS)

    Tsai, M. Y.; Morfini, G.; Szebenyi, G.; Brady, S. T.

    2000-01-01

    The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

  9. Mutant huntingtin, abnormal mitochondrial dynamics, defective axonal transport of mitochondria, and selective synaptic degeneration in Huntington's disease.

    PubMed

    Reddy, P Hemachandra; Shirendeb, Ulziibat P

    2012-02-01

    Huntington's disease (HD) is a progressive, fatal neurodegenerative disease caused by expanded polyglutamine repeats in the HD gene. HD is characterized by chorea, seizures, involuntary movements, dystonia, cognitive decline, intellectual impairment and emotional disturbances. Research into mutant huntingtin (Htt) and mitochondria has found that mutant Htt interacts with the mitochondrial protein dynamin-related protein 1 (Drp1), enhances GTPase Drp1 enzymatic activity, and causes excessive mitochondrial fragmentation and abnormal distribution, leading to defective axonal transport of mitochondria and selective synaptic degeneration. This article summarizes latest developments in HD research and focuses on the role of abnormal mitochondrial dynamics and defective axonal transport in HD neurons. This article also discusses the therapeutic strategies that decrease mitochondrial fragmentation and neuronal damage in HD.

  10. K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons II. Base influx

    PubMed Central

    1995-01-01

    We used microelectrodes to determine whether the K/HCO3 cotransporter tentatively identified in the accompanying paper (Hogan, E. M., M. A. Cohen, and W. F. Boron. 1995. Journal of General Physiology. 106:821- 844) can mediate an increase in the intracellular pH (pHi) of squid giant axons. An 80-min period of internal dialysis increased pHi to 7.7, 8.0, or 8.3; the dialysis fluid was free of K+, Na+, and Cl-. Our standard artificial seawater (ASW), which also lacked Na+, K+, and Cl-, had a pH of 8.0. Halting dialysis unmasked a slow pHi decrease. Subsequently introducing an ASW containing 437 mM K+ and 0.5% CO2/12 mM HCO3- had two effects: (a) it caused membrane potential (Vm) to become very positive, and (b) it caused a rapid pHi decrease, because of CO2 influx, followed by a slower plateau-phase pHi increase, presumably because of inward cotransport of K+ and HCO3- ("base influx"). Only extracellular Rb+ substituted for K+ in producing the plateau-phase pHi increase in the presence of CO2/HCO3-. Mean fluxes with Na+, Li+, and Cs+ were not significantly different from zero, even though Vm shifts were comparable for all monovalent cations tested. Thus, unless K+ or Rb+ (but not Na+, Li+, or Cs+) somehow activates a conductive pathway for H+, HCO3-, or both, it is unlikely that passive transport of H+, HCO3-, or both makes the major contribution to the pHi increase in the presence of K+ (or Rb+) and CO2/HCO3-. Because exposing axons to an ASW containing 437 mM K+, but no CO2/HCO3-, produced at most a slow pHi increase, K-H exchange could not make a major contribution to base influx. Introducing an ASW containing CO2/HCO3-, but no K+ also failed to elicit base influx. Because we observed base influx when the ASW and DF were free of Na+ and Cl-, and because the disulfonic stilbene derivatives SITS and DIDS failed to block base influx, Na(+)-dependent Cl-HCO3 exchange also cannot account for the results. Rather, we suggest that the most straightforward explanation for

  11. Amyloid-β oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons

    PubMed Central

    Ramser, Elisa M.; Gan, Kathlyn J.; Decker, Helena; Fan, Emily Y.; Suzuki, Matthew M.; Ferreira, Sergio T.; Silverman, Michael A.

    2013-01-01

    Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer's disease (AD). Soluble amyloid-β oligomers (AβOs), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. AβOs also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that AβOs reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3β, downstream targets of CaN, prevents BDNF transport defects induced by AβOs. We further show that AβOs induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for AβO-induced BDNF transport disruption. PMID:23783030

  12. Differential role of the low affinity neurotrophin receptor (p75) in retrograde axonal transport of the neurotrophins.

    PubMed

    Curtis, R; Adryan, K M; Stark, J L; Park, J S; Compton, D L; Weskamp, G; Huber, L J; Chao, M V; Jaenisch, R; Lee, K F

    1995-06-01

    The receptor mechanisms mediating the retrograde axonal transport of the neurotrophins have been investigated in adult rats. We show that transport of the TrkB ligands NT-4 and BDNF to peripheral neurons is dependent on the low affinity neurotrophin receptor (LNR). Pharmacological manipulation of LNR in vivo using either an anti-LNR antibody or a soluble recombinant LNR extracellular domain completely blocked retrograde transport of NT-4 and BDNF to sensory neurons, while having minimal effects on the transport of NGF in either sensory or sympathetic neurons. Furthermore, in mice with a null mutation of LNR, the transport of NT-4 and BDNF, but not NGF, was dramatically reduced. These observations demonstrate a selective role for LNR in retrograde transport of the various neurotrophins from distinct target regions in vivo.

  13. Selective interruption of axonal transport of neurofilament proteins in the visual system by beta,beta'-iminodipropionitrile (IDPN) intoxication.

    PubMed

    Parhad, I M; Griffin, J W; Hoffman, P N; Koves, J F

    1986-01-22

    Beta,beta'-iminodipropionitrile (IDPN) is an agent that produces a marked impairment in the transport of neurofilaments. Its effect on other slowly transported cytoskeletal components such as tubulin and actin is variable. Previous studies have evaluated transport of neurofilaments after IDPN intoxication in a neurofilament-rich system (sciatic motor nerves) and in a system devoid of neurofilaments (axons of the dorsal motor nucleus of the vagus). In the former, IDPN impairs the transport of tubulin and actin but to a lesser degree than it does neurofilament proteins. In the latter, tubulin and actin transport were not impaired, and neurofilament proteins were not present. In this study we evaluated the transport of the cytoskeletal components in a system with an intermediate amount of neurofilaments (the visual system). In the visual system, there is a selective and marked (50%) impairment in the transport of neurofilaments with no impairment in transport of tubulin or microtubule-associated proteins (tau group). We conclude that these different patterns of impairment in transport reflect the differences in pre-intoxication neurofilament content of the nerves examined, the effect of IDPN on the transport of the other components of slow transport being secondary to the presence of stagnated neurofilaments. This model also suggests that transport of neurofilaments can be selectively impaired without producing an effect on other major slow transport components.

  14. Myelin injury induces axonal transport impairment but not AD-like pathology in the hippocampus of cuprizone-fed mice

    PubMed Central

    Sun, Junjun; Zhou, Hong; Bai, Feng; Ren, Qingguo; Zhang, Zhijun

    2016-01-01

    Both multiple sclerosis (MS) and Alzheimer's disease (AD) are progressive neurological disorders with myelin injury and memory impairment. However, whether myelin impairment could cause AD-like neurological pathology remains unclear. To explore neurological pathology following myelin injury, we assessed cognitive function, the expression of myelin proteins, axonal transport-associated proteins, axonal structural proteins, synapse-associated proteins, tau and beta amyloid and the status of neurons, using the cuprizone mouse model of demyelination. We found the mild impairment of learning ability in cuprizone-fed mice and the decreased expression of myelin basic protein (MBP) in the hippocampus. And anti-LINGO-1 improved learning ability and partly restored MBP level. Furthermore, we also found kinesin light chain (KLC), neurofilament light chain (NFL) and neurofilament heavy chain (NF200) were declined in demyelinated hippocampus, which could be partly improved by treatment with anti-LINGO-1. However, we did not observe the increased expression of beta amyloid, hyperphosphorylation of tau and loss of neurons in demyelinated hippocampus. Our results suggest that demyelination might lead to the impairment of neuronal transport, but not cause increased level of hyperphosphorylated tau and beta amyloid. Our research demonstrates remyelination might be an effective pathway to recover the function of neuronal axons and cognition in MS. PMID:27129150

  15. A role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein

    NASA Technical Reports Server (NTRS)

    Ratner, N.; Bloom, G. S.; Brady, S. T.

    1998-01-01

    Proteins that interact with both cytoskeletal and membrane components are candidates to modulate membrane trafficking. The tumor suppressor proteins neurofibromin (NF1) and adenomatous polyposis coli (APC) both bind to microtubules and interact with membrane-associated proteins. The effects of recombinant NF1 and APC fragments on vesicle motility were evaluated by measuring fast axonal transport along microtubules in axoplasm from squid giant axons. APC4 (amino acids 1034-2844) reduced only anterograde movements, whereas APC2 (aa 1034-2130) or APC3 (aa 2130-2844) reduced both anterograde and retrograde transport. NF1 had no effect on organelle movement in either direction. Because APC contains multiple cyclin-dependent kinase (CDK) consensus phosphorylation motifs, the kinase inhibitor olomoucine was examined. At concentrations in which olomoucine is specific for cyclin-dependent kinases (5 microM), it reduced only anterograde transport, whereas anterograde and retrograde movement were both affected at concentrations at which other kinases are inhibited as well (50 microM). Both anterograde and retrograde transport also were inhibited by histone H1 and KSPXK peptides, substrates for proline-directed kinases, including CDKs. Our data suggest that CDK-like axonal kinases modulate fast anterograde transport and that other axonal kinases may be involved in modulating retrograde transport. The specific effect of APC4 on anterograde transport suggests a model in which the binding of APC to microtubules may limit the activity of axonal CDK kinase or kinases in restricted domains, thereby affecting organelle transport.

  16. A role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein

    NASA Technical Reports Server (NTRS)

    Ratner, N.; Bloom, G. S.; Brady, S. T.

    1998-01-01

    Proteins that interact with both cytoskeletal and membrane components are candidates to modulate membrane trafficking. The tumor suppressor proteins neurofibromin (NF1) and adenomatous polyposis coli (APC) both bind to microtubules and interact with membrane-associated proteins. The effects of recombinant NF1 and APC fragments on vesicle motility were evaluated by measuring fast axonal transport along microtubules in axoplasm from squid giant axons. APC4 (amino acids 1034-2844) reduced only anterograde movements, whereas APC2 (aa 1034-2130) or APC3 (aa 2130-2844) reduced both anterograde and retrograde transport. NF1 had no effect on organelle movement in either direction. Because APC contains multiple cyclin-dependent kinase (CDK) consensus phosphorylation motifs, the kinase inhibitor olomoucine was examined. At concentrations in which olomoucine is specific for cyclin-dependent kinases (5 microM), it reduced only anterograde transport, whereas anterograde and retrograde movement were both affected at concentrations at which other kinases are inhibited as well (50 microM). Both anterograde and retrograde transport also were inhibited by histone H1 and KSPXK peptides, substrates for proline-directed kinases, including CDKs. Our data suggest that CDK-like axonal kinases modulate fast anterograde transport and that other axonal kinases may be involved in modulating retrograde transport. The specific effect of APC4 on anterograde transport suggests a model in which the binding of APC to microtubules may limit the activity of axonal CDK kinase or kinases in restricted domains, thereby affecting organelle transport.

  17. Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation

    PubMed Central

    Winans, Amy M; Collins, Sean R; Meyer, Tobias

    2016-01-01

    Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon. DOI: http://dx.doi.org/10.7554/eLife.12387.001 PMID:26836307

  18. Epitope-tagged dopamine transporter knock-in mice reveal rapid endocytic trafficking and filopodia targeting of the transporter in dopaminergic axons

    PubMed Central

    Rao, Anjali; Richards, Toni L.; Simmons, Diana; Zahniser, Nancy R.; Sorkin, Alexander

    2012-01-01

    The plasma membrane dopamine (DA) transporter (DAT) is essential for reuptake of extracellular DA. DAT function in heterologous cells is regulated by subcellular targeting, endocytosis, and intracellular trafficking, but the mechanisms regulating neuronal DAT remain poorly understood. Hence, we generated a knock-in mouse expressing a hemagglutinin (HA)-epitope-tagged DAT to study endogenous transporter trafficking. Introduction of the HA tag into the second extracellular loop of mouse DAT did not perturb its expression level, distribution pattern, or substrate uptake kinetics. Live-cell fluorescence microscopy imaging using fluorescently labeled HA-specific antibody and a quantitative HA-antibody endocytosis assay demonstrated that in axons HA-DAT was primarily located in the plasma membrane and internalized mostly in growth cones and varicosities, where synaptic vesicle markers were also concentrated. Formation of varicosities was frequently preceded or accompanied by highly dynamic filopodia-like membrane protrusions. Remarkably, HA-DAT often concentrated at the tips of these filopodia. This pool of HA-DATs exhibited low lateral membrane mobility. Thus, DAT-containing filopodia may be involved in synaptogenesis in developing DA neurons. Treatment of neurons with amphetamine increased mobility of filopodial HA-DAT and accelerated HA-DAT endocytosis in axons, suggesting that chronic amphetamine may interfere with DA synapse development. Interestingly, phorbol esters did not accelerate endocytosis of axonal DAT.—Rao, A., Richards, T. L., Simmons, D., Zahniser, N. R., Sorkin, A. Epitope-tagged dopamine transporter knock-in mice reveal rapid endocytic trafficking and filopodia targeting of the transporter in dopaminergic axons. PMID:22267337

  19. Spatial and Temporal Characteristics of Normal and Perturbed Vesicle Transport

    PubMed Central

    Iacobucci, Gary J.; Rahman, Noura Abdel; Valtueña, Aida Andrades; Nayak, Tapan Kumar; Gunawardena, Shermali

    2014-01-01

    Efficient intracellular transport is essential for healthy cellular function and structural integrity, and problems in this pathway can lead to neuronal cell death and disease. To spatially and temporally evaluate how transport defects are initiated, we adapted a primary neuronal culture system from Drosophila larval brains to visualize the movement dynamics of several cargos/organelles along a 90 micron axonal neurite over time. All six vesicles/organelles imaged showed robust bi-directional motility at both day 1 and day 2. Reduction of motor proteins decreased the movement of vesicles/organelles with increased numbers of neurite blocks. Neuronal growth was also perturbed with reduction of motor proteins. Strikingly, we found that all blockages were not fixed, permanent blocks that impeded transport of vesicles as previously thought, but that some blocks were dynamic clusters of vesicles that resolved over time. Taken together, our findings suggest that non-resolving blocks may likely initiate deleterious pathways leading to death and degeneration, while resolving blocks may be benign. Therefore evaluating the spatial and temporal characteristics of vesicle transport has important implications for our understanding of how transport defects can affect other pathways to initiate death and degeneration. PMID:24878565

  20. Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain.

    PubMed

    Belmer, Arnauld; Klenowski, Paul M; Patkar, Omkar L; Bartlett, Selena E

    2017-04-01

    Serotonin neurons arise from the brainstem raphe nuclei and send their projections throughout the brain to release 5-HT which acts as a modulator of several neuronal populations. Previous electron microscopy studies in rats have morphologically determined the distribution of 5-HT release sites (boutons) in certain brain regions and have shown that 5-HT containing boutons form synaptic contacts that are either symmetric or asymmetric. In addition, 5-HT boutons can form synaptic triads with the pre- and postsynaptic specializations of either symmetrical or asymmetrical synapses. However, due to the labor intensive processing of serial sections required by electron microscopy, little is known about the neurochemical properties or the quantitative distribution of 5-HT triads within whole brain or discrete subregions. Therefore, we used a semi-automated approach that combines immunohistochemistry and high-resolution confocal microscopy to label serotonin transporter (SERT) immunoreactive axons and reconstruct in 3D their distribution within limbic brain regions. We also used antibodies against key pre- (synaptophysin) and postsynaptic components of excitatory (PSD95) or inhibitory (gephyrin) synapses to (1) identify putative 5-HTergic boutons within SERT immunoreactive axons and, (2) quantify their close apposition to neurochemical excitatory or inhibitory synapses. We provide a 5-HTergic axon density map and have determined the ratio of synaptic triads consisting of a 5-HT bouton in close proximity to either neurochemical excitatory or inhibitory synapses within different limbic brain areas. The ability to model and map changes in 5-HTergic axonal density and the formation of triadic connectivity within whole brain regions using this rapid and quantitative approach offers new possibilities for studying neuroplastic changes in the 5-HTergic pathway.

  1. Retrograde axonal transport of LIF is increased by peripheral nerve injury: correlation with increased LIF expression in distal nerve.

    PubMed

    Curtis, R; Scherer, S S; Somogyi, R; Adryan, K M; Ip, N Y; Zhu, Y; Lindsay, R M; DiStefano, P S

    1994-01-01

    Leukemia inhibitory factor (LIF) is a cytokine that affects the survival and differentiation of certain neuronal populations in vitro. To identify LIF-responsive neurons in the adult rat, we have demonstrated retrograde axonal transport of 125I-LIF to sensory and motor neurons. The accumulation of 125I-LIF by both cell types was significantly increased by prior sciatic nerve crush. Retrograde transport of 125I-LIF was inhibited by excess unlabeled LIF but not by related cytokines, indicating a specific receptor-mediated mechanism. Northern blot analysis revealed LIF expression in peripheral nerve that was increased in distal segments after axotomy. The correlation between LIF expression and increased retrograde transport following injury suggests that LIF plays a role in peripheral nerve regeneration.

  2. Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: Implications for chemotherapy-induced peripheral neuropathy

    PubMed Central

    LaPointe, Nichole E.; Morfini, Gerardo; Brady, Scott T.; Feinstein, Stuart C.; Wilson, Leslie; Jordan, Mary Ann

    2014-01-01

    Chemotherapy-induced peripheral neuropathy (CIPN) is a serious, painful and dose-limiting side effect of cancer drugs that target microtubules. The mechanisms underlying the neuronal damage are unknown, but may include disruption of fast axonal transport, an essential microtubule-based process that moves cellular components over long distances between neuronal cell bodies and nerve terminals. This idea is supported by the “dying back” pattern of degeneration observed in CIPN, and by the selective vulnerability of sensory neurons bearing the longest axonal projections. In this study, we test the hypothesis that microtubule-targeting drugs disrupt fast axonal transport using vesicle motility assays in isolated squid axoplasm and a cell-free microtubule gliding assay with defined components. We compare four clinically-used drugs, eribulin, vincristine, paclitaxel and ixabepilone. Of these, eribulin is associated with a relatively low incidence of severe neuropathy, while vincristine has a relatively high incidence. In vesicle motility assays, we found that all four drugs inhibited anterograde (conventional kinesin-dependent) fast axonal transport, with the potency being vincristine = ixabepilone > paclitaxel = eribulin. Interestingly, eribulin and paclitaxel did not inhibit retrograde (cytoplasmic dynein-dependent) fast axonal transport, in contrast to vincristine and ixabepilone. Similarly, vincristine and ixabepilone both exerted significant inhibitory effects in an in vitro microtubule gliding assay consisting of recombinant kinesin (kinesin-1) and microtubules composed of purified bovine brain tubulin, whereas paclitaxel and eribulin had negligible effects. Our results suggest that (i) inhibition of microtubule-based fast axonal transport may be a significant contributor to neurotoxicity induced by microtubule-targeting drugs, and (ii) that individual microtubule-targeting drugs affect fast axonal transport through different mechanisms. PMID:23711742

  3. Axonal transport of neurotrophins by visceral afferent and efferent neurons of the vagus nerve of the rat.

    PubMed

    Helke, C J; Adryan, K M; Fedorowicz, J; Zhuo, H; Park, J S; Curtis, R; Radley, H E; Distefano, P S

    1998-03-30

    The receptor-mediated axonal transport of [125I]-labeled neurotrophins by afferent and efferent neurons of the vagus nerve was determined to predict the responsiveness of these neurons to neurotrophins in vivo. [125I]-labeled neurotrophins were administered to the proximal stump of the transected cervical vagus nerve of adult rats. Vagal afferent neurons retrogradely transported [125I]neurotrophin-3 (NT-3), [125I]nerve growth factor (NGF), and [125I]neurotrophin-4 (NT-4) to perikarya in the ipsilateral nodose ganglion, and transganglionically transported [125I]NT-3, [125I]NGF, and [125I]NT-4 to the central terminal field, the nucleus tractus solitarius (NTS). Vagal afferent neurons showed minimal accumulation of [125I]brain-derived neurotrophic factor (BDNF). In contrast, efferent (parasympathetic and motor) neurons located in the dorsal motor nucleus of the vagus and nucleus ambiguus retrogradely transported [125I]BDNF, [125I]NT-3, and [125I]NT-4, but not [125I]NGF. The receptor specificity of neurotrophin transport was examined by applying [125I]-labeled neurotrophins with an excess of unlabeled neurotrophins. The retrograde transport of [125I]NT-3 to the nodose ganglion was reduced by NT-3 and by NGF, and the transport of [125I]NGF was reduced only by NGF, whereas the transport of [125I]NT-4 was significantly reduced by each of the neurotrophins. The competition profiles for the transport of NT-3 and NGF are consistent with the presence of TrkA and TrkC and the absence of TrkB in the nodose ganglion, whereas the profile for NT-4 suggests a p75 receptor-mediated transport mechanism. The transport profiles of neurotrophins by efferent vagal neurons in the dorsal motor nucleus of the vagus and nucleus ambiguus are consistent with the presence of TrkB and TrkC, but not TrkA, in these nuclei. These observations describe the unique receptor-mediated axonal transport of neurotrophins in adult vagal afferent and efferent neurons and thus serve as a template to discern

  4. Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons

    PubMed Central

    1992-01-01

    Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers. PMID:1383234

  5. Dual-specificity phosphatase 26 (DUSP26) stimulates Aβ42 generation by promoting amyloid precursor protein axonal transport during hypoxia.

    PubMed

    Jung, Sunmin; Nah, Jihoon; Han, Jonghee; Choi, Seon-Guk; Kim, Hyunjoo; Park, Jaesang; Pyo, Ha-Kyung; Jung, Yong-Keun

    2016-06-01

    Amyloid beta peptide (Aβ) is a pathological hallmark of Alzheimer's disease (AD) and is generated through the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. Hypoxia is a known risk factor for AD and stimulates Aβ generation by γ-secretase; however, the underlying mechanisms remain unclear. In this study, we showed that dual-specificity phosphatase 26 (DUSP26) regulates Aβ generation through changes in subcellular localization of the γ-secretase complex and its substrate C99 under hypoxic conditions. DUSP26 was identified as a novel γ-secretase regulator from a genome-wide functional screen using a cDNA expression library. The phosphatase activity of DUSP26 was required for the increase in Aβ42 generation through γ-secretase, but this regulation did not affect the amount of the γ-secretase complex. Interestingly, DUSP26 induced the accumulation of C99 in the axons by stimulating anterograde transport of C99-positive vesicles. Additionally, DUSP26 induced c-Jun N-terminal kinase (JNK) activation for APP processing and axonal transport of C99. Under hypoxic conditions, DUSP26 expression levels were elevated together with JNK activation, and treatment with JNK inhibitor SP600125, or the DUSP26 inhibitor NSC-87877, reduced hypoxia-induced Aβ generation by diminishing vesicle trafficking of C99 to the axons. Finally, we observed enhanced DUSP26 expression and JNK activation in the hippocampus of AD patients. Our results suggest that DUSP26 mediates hypoxia-induced Aβ generation through JNK activation, revealing a new regulator of γ-secretase-mediated APP processing under hypoxic conditions. We propose the role of phosphatase dual-specificity phosphatase 26 (DUSP26) in the selective regulation of Aβ42 production in neuronal cells under hypoxic stress. Induction of DUSP26 causes JNK-dependent shift in the subcellular localization of γ-secretase and C99 from the cell body to axons for Aβ42 generation. These findings provide a

  6. TRiC subunits enhance BDNF axonal transport and rescue striatal atrophy in Huntington’s disease

    PubMed Central

    Zhao, Xiaobei; Chen, Xu-Qiao; Han, Eugene; Hu, Yue; Paik, Paul; Ding, Zhiyong; Overman, Julia; Lau, Alice L.; Shahmoradian, Sarah H.; Chiu, Wah; Thompson, Leslie M.; Wu, Chengbiao; Mobley, William C.

    2016-01-01

    Corticostriatal atrophy is a cardinal manifestation of Huntington’s disease (HD). However, the mechanism(s) by which mutant huntingtin (mHTT) protein contributes to the degeneration of the corticostriatal circuit is not well understood. We recreated the corticostriatal circuit in microfluidic chambers, pairing cortical and striatal neurons from the BACHD model of HD and its WT control. There were reduced synaptic connectivity and atrophy of striatal neurons in cultures in which BACHD cortical and striatal neurons were paired. However, these changes were prevented if WT cortical neurons were paired with BACHD striatal neurons; synthesis and release of brain-derived neurotrophic factor (BDNF) from WT cortical axons were responsible. Consistent with these findings, there was a marked reduction in anterograde transport of BDNF in BACHD cortical neurons. Subunits of the cytosolic chaperonin T-complex 1 (TCP-1) ring complex (TRiC or CCT for chaperonin containing TCP-1) have been shown to reduce mHTT levels. Both CCT3 and the apical domain of CCT1 (ApiCCT1) decreased the level of mHTT in BACHD cortical neurons. In cortical axons, they normalized anterograde BDNF transport, restored retrograde BDNF transport, and normalized lysosomal transport. Importantly, treating BACHD cortical neurons with ApiCCT1 prevented BACHD striatal neuronal atrophy by enhancing release of BDNF that subsequently acts through tyrosine receptor kinase B (TrkB) receptor on striatal neurons. Our findings are evidence that TRiC reagent-mediated reductions in mHTT enhanced BDNF delivery to restore the trophic status of BACHD striatal neurons. PMID:27601642

  7. Distal retinal ganglion cell axon transport loss and activation of p38 MAPK stress pathway following VEGF-A antagonism

    PubMed Central

    Foxton, R; Osborne, A; Martin, K R; Ng, Y-S; Shima, D T

    2016-01-01

    There is increasing evidence that VEGF-A antagonists may be detrimental to neuronal health following ocular administration. Here we investigated firstly the effects of VEGF-A neutralization on retinal neuronal survival in the Ins2Akita diabetic and JR5558 spontaneous choroidal neovascularization (CNV) mice, and then looked at potential mechanisms contributing to cell death. We detected elevated apoptosis in the ganglion cell layer in both these models following VEGF-A antagonism, indicating that even when vascular pathologies respond to treatment, neurons are still vulnerable to reduced VEGF-A levels. We observed that retinal ganglion cells (RGCs) seemed to be the cells most susceptible to VEGF-A antagonism, so we looked at anterograde transport in these cells, due to their long axons requiring optimal protein and organelle trafficking. Using cholera toxin B-subunit tracer studies, we found a distal reduction in transport in the superior colliculus following VEGF-A neutralization, which occurred prior to net RGC loss. This phenomenon of distal transport loss has been described as a feature of early pathological changes in glaucoma, Alzheimer's and Parkinson's disease models. Furthermore, we observed increased phosphorylation of p38 MAPK and downstream Hsp27 stress pathway signaling in the retinas from these experiments, potentially providing a mechanistic explanation for our findings. These experiments further highlight the possible risks of using VEGF-A antagonists to treat ocular neovascular disease, and suggest that VEGF-A may contribute to the maintenance and function of axonal transport in neurons of the retina. PMID:27148685

  8. Sexual divergence in microtubule function: the novel intranasal microtubule targeting SKIP normalizes axonal transport and enhances memory.

    PubMed

    Amram, N; Hacohen-Kleiman, G; Sragovich, S; Malishkevich, A; Katz, J; Touloumi, O; Lagoudaki, R; Grigoriadis, N C; Giladi, E; Yeheskel, A; Pasmanik-Chor, M; Jouroukhin, Y; Gozes, I

    2016-10-01

    Activity-dependent neuroprotective protein (ADNP), essential for brain formation, is a frequent autism spectrum disorder (ASD)-mutated gene. ADNP associates with microtubule end-binding proteins (EBs) through its SxIP motif, to regulate dendritic spine formation and brain plasticity. Here, we reveal SKIP, a novel four-amino-acid peptide representing an EB-binding site, as a replacement therapy in an outbred Adnp-deficient mouse model. We discovered, for the first time, axonal transport deficits in Adnp(+/-) mice (measured by manganese-enhanced magnetic resonance imaging), with significant male-female differences. RNA sequencing evaluations showed major age, sex and genotype differences. Function enrichment and focus on major gene expression changes further implicated channel/transporter function and the cytoskeleton. In particular, a significant maturation change (1 month-five months) was observed in beta1 tubulin (Tubb1) mRNA, only in Adnp(+/+) males, and sex-dependent increase in calcium channel mRNA (Cacna1e) in Adnp(+/+) males compared with females. At the protein level, the Adnp(+/-) mice exhibited impaired hippocampal expression of the calcium channel (voltage-dependent calcium channel, Cacnb1) as well as other key ASD-linked genes including the serotonin transporter (Slc6a4), and the autophagy regulator, BECN1 (Beclin1), in a sex-dependent manner. Intranasal SKIP treatment normalized social memory in 8- to 9-month-old Adnp(+/-)-treated mice to placebo-control levels, while protecting axonal transport and ameliorating changes in ASD-like gene expression. The control, all d-amino analog D-SKIP, did not mimic SKIP activity. SKIP presents a novel prototype for potential ASD drug development, a prevalent unmet medical need.

  9. Predicting myelinated axon activation using spatial characteristics of the extracellular field

    PubMed Central

    Peterson, EJ; Izad, O; Tyler, DJ

    2011-01-01

    Computation time required for modeling the nonlinear response of an axon to an applied electric field is a significant limitation to optimizing a large number of neural interface design parameters through use of advanced computer algorithms. This paper introduces two methods of predicting axon activation that incorporate a threshold that includes the magnitude of the extracellular potential to achieve increased accuracy over previous computationally efficient methods. Each method employs the use of a modified driving function that includes the second spatial difference of the applied extracellular voltage to predict the electrical excitation of a nerve. The first method uses the second spatial difference taken at a single node of Ranvier, while the second uses a weighted sum of the second spatial differences taken at all nodes of Ranvier. This study quantifies prediction accuracy for cases with single and multiple point source stimulating electrodes. While both new methods address the major criticism of linearized prediction models, the weighted sum method provides the most robust response across single and multiple point sources. These methods eimprove prediction of axon activation based on properties of the applied field in a computationally efficient manner. PMID:21750371

  10. Increased expression of reticulon 3 in neurons leads to reduced axonal transport of β site amyloid precursor protein-cleaving enzyme 1.

    PubMed

    Deng, Minzi; He, Wanxia; Tan, Ya; Han, Hailong; Hu, Xiangyou; Xia, Kun; Zhang, Zhuohua; Yan, Riqiang

    2013-10-18

    BACE1 is the sole enzyme responsible for cleaving amyloid precursor protein at the β-secretase site, and this cleavage initiates the generation of β-amyloid peptide (Aβ). Because amyloid precursor protein is predominantly expressed by neurons and deposition of Aβ aggregates in the human brain is highly correlated with the Aβ released at axonal terminals, we focused our investigation of BACE1 localization on the neuritic region. We show that BACE1 was not only enriched in the late Golgi, trans-Golgi network, and early endosomes but also in both axons and dendrites. BACE1 was colocalized with the presynaptic vesicle marker synaptophysin, indicating the presence of BACE1 in synapses. Because the excessive release of Aβ from synapses is attributable to an increase in amyloid deposition, we further explored whether the presence of BACE1 in synapses was regulated by reticulon 3 (RTN3), a protein identified previously as a negative regulator of BACE1. We found that RTN3 is not only localized in the endoplasmic reticulum but also in neuritic regions where no endoplasmic reticulum-shaping proteins are detected, implicating additional functions of RTN3 in neurons. Coexpression of RTN3 with BACE1 in cultured neurons was sufficient to reduce colocalization of BACE1 with synaptophysin. This reduction correlated with decreased anterograde transport of BACE1 in axons in response to overexpressed RTN3. Our results in this study suggest that altered RTN3 levels can impact the axonal transport of BACE1 and demonstrate that reducing axonal transport of BACE1 in axons is a viable strategy for decreasing BACE1 in axonal terminals and, perhaps, reducing amyloid deposition.

  11. Restoring axonal localization and transport of transmembrane receptors to promote repair within the injured CNS: a critical step in CNS regeneration

    PubMed Central

    Forbes, Lindsey H.; Andrews, Melissa R.

    2017-01-01

    Each neuronal subtype is distinct in how it develops, responds to environmental cues, and whether it is capable of mounting a regenerative response following injury. Although the adult central nervous system (CNS) does not regenerate, several experimental interventions have been trialled with successful albeit limited instances of axonal repair. We highlight here some of these approaches including extracellular matrix (ECM) modification, cellular grafting, gene therapy-induced replacement of proteins, as well as application of biomaterials. We also review the recent report demonstrating the failure of axonal localization and transport of growth-promoting receptors within certain classes of mature neurons. More specifically, we discuss an inability of integrin receptors to localize within the axonal compartment of mature motor neurons such as in the corticospinal and rubrospinal tracts, whereas in immature neurons of those pathways and in mature sensory tracts such as in the optic nerve and dorsal column pathways these receptors readily localize within axons. Furthermore we assert that this failure of axonal localization contributes to the intrinsic inability of axonal regeneration. We conclude by highlighting the necessity for both combined therapies as well as a targeted approach specific to both age and neuronal subtype will be required to induce substantial CNS repair. PMID:28250734

  12. Long-distance Axonal Transport of AAV9 Is Driven by Dynein and Kinesin-2 and Is Trafficked in a Highly Motile Rab7-positive Compartment

    PubMed Central

    Castle, Michael J; Perlson, Eran; Holzbaur, Erika LF; Wolfe, John H

    2014-01-01

    Adeno-associated virus (AAV) vectors can move along axonal pathways after brain injection, resulting in transduction of distal brain regions. This can enhance the spread of therapeutic gene transfer and improve treatment of neurogenetic disorders that require global correction. To better understand the underlying cellular mechanisms that drive AAV trafficking in neurons, we investigated the axonal transport of dye-conjugated AAV9, utilizing microfluidic primary neuron cultures that isolate cell bodies from axon termini and permit independent analysis of retrograde and anterograde axonal transport. After entry, AAV was trafficked into nonmotile early and recycling endosomes, exocytic vesicles, and a retrograde-directed late endosome/lysosome compartment. Rab7-positive late endosomes/lysosomes that contained AAV were highly motile, exhibiting faster retrograde velocities and less pausing than Rab7-positive endosomes without virus. Inhibitor experiments indicated that the retrograde transport of AAV within these endosomes is driven by cytoplasmic dynein and requires Rab7 function, whereas anterograde transport of AAV is driven by kinesin-2 and exhibits unusually rapid velocities. Furthermore, increasing AAV9 uptake by neuraminidase treatment significantly enhanced virus transport in both directions. These findings provide novel insights into AAV trafficking within neurons, which should enhance progress toward the utilization of AAV for improved distribution of transgene delivery within the brain. PMID:24100640

  13. Lissencephaly-1 dependent axonal retrograde transport of L1-type CAM Neuroglian in the adult drosophila central nervous system

    PubMed Central

    Börner, Jana; Slipchuk, Olesya; Kakad, Priyanka; Lee, LaTasha H.; Qureshi, Aater; Pielage, Jan

    2017-01-01

    Here, we established the Drosophila Giant Fiber neurons (GF) as a novel model to study axonal trafficking of L1-type Cell Adhesion Molecules (CAM) Neuroglian (Nrg) in the adult CNS using live imaging. L1-type CAMs are well known for their importance in nervous system development and we previously demonstrated a role for Nrg in GF synapse formation. However, in the adult they have also been implicated in synaptic plasticity and regeneration. In addition, to its canonical role in organizing cytoskeletal elements at the plasma membrane, vertebrate L1CAM has also been shown to regulate transcription indirectly as well as directly via its import to the nucleus. Here, we intend to determine if the sole L1CAM homolog Nrg is retrogradley transported and thus has the potential to relay signals from the synapse to the soma. Live imaging of c-terminally tagged Nrg in the GF revealed that there are at least two populations of retrograde vesicles that differ in speed, and either move with consistent or varying velocity. To determine if endogenous Nrg is retrogradely transported, we inhibited two key regulators, Lissencephaly-1 (Lis1) and Dynactin, of the retrograde motor protein Dynein. Similar to previously described phenotypes for expression of poisonous subunits of Dynactin, we found that developmental knock down of Lis1 disrupted GF synaptic terminal growth and that Nrg vesicles accumulated inside the stunted terminals in both mutant backgrounds. Moreover, post mitotic Lis1 knock down in mature GFs by either RNAi or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) induced mutations, resulted in normal length terminals with fully functional GF synapses which also exhibited severe accumulation of endogenous Nrg vesicles. Thus, our data suggests that accumulation of Nrg vesicles is due to failure of retrograde transport rather than a failure of terminal development. Together with the finding that post mitotic knock down of Lis1 also disrupted retrograde

  14. An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: Immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L).

    PubMed

    Gerfen, Charles R; Sawchenko, Paul E

    2016-08-15

    A new neuroanatomical method for tracing connections in the central nervous system based on the anterograde axonal transport of the kidney bean lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L) is described. The method, for which a detailed protocol is presented, offers several advantages over present techniques. First, when the lectin is delivered iontophoretically, PHA-L injection sites as small as 50-200μm in diameter can be produced, and are clearly demarcated since the neurons within the labeled zone are completely filled. Second, many morphological features of such filled neurons are clearly demonstrated including their cell bodies, axons, dendritic arbors and even dendritic spines. Third, there is some evidence to suggest that only the neurons at the injection site that are filled transport demonstrable amounts of the tracer, raising the possibility that the effective injection site can be defined quite precisely. Fourth, even with the most restricted injections, the morphology of the labeled axons and axon terminals is clearly demonstrated; this includes boutons en passant, fine collateral branches, and various terminal specialization, all of which can be visualized as well as in the best rapid Golgi preparations. Fifth, when introduced iontophoretically, PHA-L appears to be transported preferentially in the anterograde direction; only rarely is it transported retrogradely. Sixth, PHA-L does not appear to be taken up and transported effectively by fibers of passage. Seventh, there is no discernible degradation of the transported PHA-L with survival times of up to 17 days. Finally, since the transported marker can be demonstrated with either peroxidase or fluorescent antibody techniques, it may be used in conjunction with other neuroanatomical methods. For example, double anterograde labeling experiments can be done using the autoradiographic method along with immunoperoxidase localization of PHA-L, and the retrogradely transported fluorescent dyes can be

  15. Distribution of pressure-induced fast axonal transport abnormalities in primate optic nerve. An autoradiographic study.

    PubMed

    Radius, R L

    1981-07-01

    The distribution of transport abnormalities in primate optic nerve from eyes subjected to five hours of pressure elevation (perfusion pressure of 35 mm Hg) was studied. Tissue autoradiography and electron microscopy were used to localize regions of the lamina cribrosa with increased transport interruption. A preferential involvement by this transport abnormality involved the superior, temporal, and inferior portions, to the exclusion of the nasal portion, of the optic nerve head. This observation supports the hypothesis that transport interruption seen in this model may be pertinent to the study of clinical glaucomatous neuropathy.

  16. Disruption of the NF-H Gene Increases Axonal Microtubule Content and Velocity of Neurofilament Transport: Relief of Axonopathy Resulting from the Toxin β,β′-Iminodipropionitrile

    PubMed Central

    Zhu, Qinzhang; Lindenbaum, Michael; Levavasseur, Françoise; Jacomy, Hélène; Julien, Jean-Pierre

    1998-01-01

    To investigate the role of the neurofilament heavy (NF-H) subunit in neuronal function, we generated mice bearing a targeted disruption of the gene coding for the NF-H subunit. Surprisingly, the lack of NF-H subunits had little effect on axonal calibers and electron microscopy revealed no significant changes in the number and packing density of neurofilaments made up of only the neurofilament light (NF-L) and neurofilament medium (NF-M) subunits. However, our analysis of NF-H knockout mice revealed an ∼2.4-fold increase of microtubule density in their large ventral root axons. This finding was further corroborated by a corresponding increase in the ratio of assembled tubulin to NF-L protein in insoluble cytoskeletal preparations from the sciatic nerve. Axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed an increased transport velocity of newly synthesized NF-L and NF-M proteins in motor axons of NF-H knockout mice. When treated with β,β′-iminodipropionitrile (IDPN), a neurotoxin that segregates microtubules and retards neurofilament transport, mice heterozygous or homozygous for the NF-H null mutation did not develop neurofilamentous swellings in motor neurons, unlike normal mouse littermates. These results indicate that the NF-H subunit is a key mediator of IDPN-induced axonopathy. PMID:9763430

  17. Motor deficit in a tauopathy model is induced by disturbances of axonal transport leading to dying-back degeneration and denervation of neuromuscular junctions.

    PubMed

    Audouard, Emilie; Van Hees, Laura; Suain, Valérie; Yilmaz, Zehra; Poncelet, Luc; Leroy, Karelle; Brion, Jean-Pierre

    2015-10-01

    Several neurodegenerative diseases are characterized by both cognitive and motor deficits associated with accumulation of tau aggregates in brain, brainstem, and spinal cord. The Tg30 murine tauopathy model expresses a human tau protein bearing two frontotemporal dementia with Parkinsonism linked to chromosome 17 pathogenic mutations and develops a severe motor deficit and tau aggregates in brain and spinal cord. To investigate the origin of this motor deficit, we analyzed the age-dependent innervation status of the neuromuscular junctions and mutant tau expression in Tg30 mice. The human transgenic tau was detected from postnatal day 7 onward in motoneurons, axons in the sciatic nerve, and axon terminals of the neuromuscular junctions. The development and maturation of neuromuscular junctions were not disrupted in Tg30 mice, but their maintenance was disturbed in adult Tg30 mice, resulting in a progressive and severe muscle denervation. This muscle denervation was associated with early electrophysiological signs of muscle spontaneous activities and histological signs of muscle degeneration. Early loss of synaptic vesicles in axon terminals preceding motor deficits, accumulation of Gallyas-positive aggregates, and cathepsin-positive vesicular clusters in axons in the sciatic nerve suggest that this denervation results from disturbances of axonal transport. This physiopathological mechanism might be responsible for motor signs observed in some human tauopathies, and for synaptic dysfunction resulting from alterations at the presynaptic level in these diseases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  18. Nebula/DSCR1 Upregulation Delays Neurodegeneration and Protects against APP-Induced Axonal Transport Defects by Restoring Calcineurin and GSK-3β Signaling

    PubMed Central

    Shaw, Jillian L.; Chang, Karen T.

    2013-01-01

    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. PMID:24086147

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

    PubMed

    Shaw, Jillian L; Chang, Karen T

    2013-01-01

    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.

  20. Retrograde axonal transport of ciliary neurotrophic factor is increased by peripheral nerve injury.

    PubMed

    Curtis, R; Adryan, K M; Zhu, Y; Harkness, P J; Lindsay, R M; DiStefano, P S

    1993-09-16

    Ciliary neurotrophic factor (CNTF) promotes the survival of several populations of neurons, including sensory and motor neurons. Although CNTF is abundant in adult sciatic nerve, the mature protein lacks a signal sequence and is not secreted; therefore, it has been proposed to act as a lesion factor. The identification of a functional CNTF receptor revealed ligand-specific phosphorylation cascades and gene induction. However, it is not clear how these signal-transducing events are elicited in neuronal cell bodies that may be distant from the source of CNTF. We report here that CNTF can be retrogradely transported by adult sensory neurons. More importantly, sensory and motor neurons both show greatly increased transport of CNTF following peripheral nerve lesion. Axotomy-induced increases in retrograde transport of neurotrophic factors may be an important response of neuronal cell bodies during regeneration.

  1. Synthesis, axonal transport, and turnover of the high molecular weight microtubule-associated protein MAP 1A in mouse retinal ganglion cells: tubulin and MAP 1A display distinct transport kinetics.

    PubMed

    Nixon, R A; Fischer, I; Lewis, S E

    1990-02-01

    Microtubule-associated proteins (MAPs) in neurons establish functional associations with microtubules, sometimes at considerable distances from their site of synthesis. In this study we identified MAP 1A in mouse retinal ganglion cells and characterized for the first time its in vivo dynamics in relation to axonally transported tubulin. A soluble 340-kD polypeptide was strongly radiolabeled in ganglion cells after intravitreal injection of [35S]methionine or [3H]proline. This polypeptide was identified as MAP 1A on the basis of its co-migration on SDS gels with MAP 1A from brain microtubules; its co-assembly with microtubules in the presence of taxol or during cycles of assembly-disassembly; and its cross-reaction with well-characterized antibodies against MAP 1A in immunoblotting and immunoprecipitation assays. Glial cells of the optic nerve synthesized considerably less MAP 1A than neurons. The axoplasmic transport of MAP 1A differed from that of tubulin. Using two separate methods, we observed that MAP 1A advanced along optic axons at a rate of 1.0-1.2 mm/d, a rate typical of the Group IV (SCb) phase of transport, while tubulin moved 0.1-0.2 mm/d, a group V (SCa) transport rate. At least 13% of the newly synthesized MAP 1A entering optic axons was incorporated uniformly along axons into stationary axonal structures. The half-residence time of stationary MAP 1A in axons (55-60 d) was 4.6 times longer than that of MAP 1A moving in Group IV, indicating that at least 44% of the total MAP 1A in axons is stationary. These results demonstrate that cytoskeletal proteins that become functionally associated with each other in axons may be delivered to these sites at different transport rates. Stable associations between axonal constituents moving at different velocities could develop when these elements leave the transport vector and incorporate into the stationary cytoskeleton.

  2. Lithium reverses behavioral and axonal transport-related changes associated with ANK3 bipolar disorder gene disruption.

    PubMed

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

    2017-03-01

    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.

  3. Analysis of the apparent biphasic axonal transport kinetics of fucosylated glycoproteins

    SciTech Connect

    Goodrum, J.F.; Morell, P.

    1984-07-01

    Following intraocular injection of (/sup 3/H)fucose, the accumulation of transported radioactivity arriving at the superior colliculus peaks within a few hours and decays with a time course of hours. Then, over a period of several days, radioactivity again accumulates at the superior colliculus and then decays with a half-life of days. Such data have been interpreted as evidence for both a group of rapidly released, rapidly transported glycoproteins (first peak) and a group of slowly released but rapidly transported glycoproteins (second peak). This supposition was investigated by studying in more detail the metabolism of some individual fucosylated proteins in both the retina and superior colliculus. It was noted that much of the radioactivity incorporated in fucosylated glycoproteins at the retina was rapidly metabolized, while the remainder of the fucosylated moieties had a metabolic half-life on the order of days. In other experiments (/sup 35/S)methionine was injected intraocularly, the metabolism in the retina was examined and a study was made of the kinetics of transport to the superior colliculus of the peptide backbone of these same individual proteins. In contrast to the two waves of accumulation of radioactivity from (/sup 3/H)fucose, accumulation of radioactivity of the peptide backbone of the same glycoproteins was monophasic. The author's explanation of these data involves the presence of two types of fucose moieties on the peptides. One group of fucose moieties is labile and is lost from the peptide backbone over a period of hours. Other fucose moieties are approximately as metabolically stable as the peptide backbones to which they are attached. The actual peptide backbones of the glycoproteins are committed to rapid transport over a period of several days.

  4. Endogenous axoplasmic proteins and proteins containing nuclear localization signal sequences use the retrograde axonal transport/nuclear import pathway in Aplysia neurons.

    PubMed

    Schmied, R; Huang, C C; Zhang, X P; Ambron, D A; Ambron, R T

    1993-09-01

    When the nuclear localization signal peptide (sp) of the SV 40 large T antigen was coupled to human serum albumin (HSA), rhodaminated (r), and microinjected into axons of Aplysia neurons in vitro, the rHSA-sp was conveyed through the axon to the cell body and then into the nucleus (Ambron et al., 1992). But since rHSA-sp is an artificial construct, we needed to determine whether naturally occurring nuclear proteins use this pathway. We therefore injected calf thymus histone H-1 and Xenopus oocyte nucleoplasmin into axons. By 3 hr both were retrogradely transported and targeted into the nucleus, though histone H-1 less efficiently than rHSA-sp or nucleoplasmin. In contrast, neither rHSA, nor rHSA conjugated to a peptide with a random distribution of basic amino acids, was transported or imported. To see if proteins that use the pathway remain intact, we coupled sp to HRP. When injected into varicosities, the HRP-sp was transported/imported to the nucleus, where it was enzymatically active. A key issue was to determine whether endogenous proteins use this pathway. Consequently, axoplasm was extruded from Aplysia nerves and the proteins were fractionated by size. SDS-PAGE and Western blots showed that two fractions contained proteins that were recognized by an affinity-purified antibody to sp: fraction 3 included sp83, and fraction 4 contained sp75. In addition, these two proteins were found in nuclei isolated from neurons. To assess transport, the total proteins in the fractions were rhodaminated and injected into varicosities. Fraction 3, but not fraction 4, contained protein that was transported through the axon to the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Traveling wave to a reaction-hyperbolic system for axonal transport

    NASA Astrophysics Data System (ADS)

    Huang, Feimin; Li, Xing; Zhang, Yinglong

    2017-07-01

    In this paper, we study a class of nonlinear reaction-hyperbolic systems modeling the neuronal signal transfer in neuroscience. This reaction-hyperbolic system can be regarded as n × n (n ≥ 2) hyperbolic system with relaxation. We first prove the existence of traveling wave by Gershgorin circle theorem and mathematically describe the neuronal signal transport. Then for a special case n = 2, we show the traveling wave is nonlinearly stable, and obtain the convergence rate simultaneously by a weighted estimate.

  6. JIP3 regulates neuronal radial migration by mediating TrkB axonal anterograde transport in the developing cerebral cortex.

    PubMed

    Ma, Huixian; Yu, Hui; Li, Ting; Zhao, Yan; Hou, Ming; Chen, Zheyu; Wang, Yue; Sun, Tao

    2017-04-15

    Radial migration is essential for the precise lamination and the coordinated function of the cerebral cortex. However, the molecular mechanisms for neuronal radial migration are not clear. Here, we report that c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) is highly expressed in the brain of embryonic mice and essential for radial migration. Knocking down JIP3 by in utero electroporation specifically perturbs the radial migration of cortical neurons but has no effect on neurogenesis and neuronal differentiation. Furthermore, we illustrate that JIP3 knockdown delays but does not block the migration of cortical neurons by investigating the distribution of neurons with JIP3 knocked down in the embryo and postnatal mouse. Finally, we find that JIP3 regulates cortical neuronal migration by mediating TrkB axonal anterograde transport during brain development. These findings deepen our understanding of the regulation of neuronal development by JIP3 and provide us a novel view on the regulating mechanisms of neuronal radial migration. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. MAPT Genetic Variation and Neuronal Maturity Alter Isoform Expression Affecting Axonal Transport in iPSC-Derived Dopamine Neurons.

    PubMed

    Beevers, Joel E; Lai, Mang Ching; Collins, Emma; Booth, Heather D E; Zambon, Federico; Parkkinen, Laura; Vowles, Jane; Cowley, Sally A; Wade-Martins, Richard; Caffrey, Tara M

    2017-08-08

    The H1 haplotype of the microtubule-associated protein tau (MAPT) locus is genetically associated with neurodegenerative diseases, including Parkinson's disease (PD), and affects gene expression and splicing. However, the functional impact on neurons of such expression differences has yet to be fully elucidated. Here, we employ extended maturation phases during differentiation of induced pluripotent stem cells (iPSCs) into mature dopaminergic neuronal cultures to obtain cultures expressing all six adult tau protein isoforms. After 6 months of maturation, levels of exon 3+ and exon 10+ transcripts approach those of adult brain. Mature dopaminergic neuronal cultures display haplotype differences in expression, with H1 expressing 22% higher levels of MAPT transcripts than H2 and H2 expressing 2-fold greater exon 3+ transcripts than H1. Furthermore, knocking down adult tau protein variants alters axonal transport velocities in mature iPSC-derived dopaminergic neuronal cultures. This work links haplotype-specific MAPT expression with a biologically functional outcome relevant for PD. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. 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.

    PubMed

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

    2017-06-01

    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

  9. Sequence determinants of the Caenhorhabditis elegans dopamine transporter dictating in vivo axonal export and synaptic localization.

    PubMed

    Robinson, Sarah B; Hardaway, J Andrew; Hardie, Shannon L; Wright, Jane; Glynn, Ryan M; Bermingham, Daniel P; Han, Qiao; Sturgeon, Sarah M; Freeman, Phyllis; Blakely, Randy D

    2017-01-01

    The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies. Loss of function mutations in the gene encoding the Caenhorhabditis elegans DAT (dat-1) produces rapid immobility when animals are placed in water, a phenotype termed swimming-induced paralysis (Swip). The ability of a DA neuron-expressed, GFP-tagged DAT-1 fusion protein (GFP::DAT-1) to localize to synapses and rescue Swip in these animals provides a facile approach to define sequences supporting DAT somatic export and function in vivo. In prior studies, we found that truncation of the last 25 amino acids of the DAT-1 C-terminus (Δ25) precludes Swip rescue, supported by a deficit in GFP::DAT-1 synaptic localization. Here, we further defined the elements within Δ25 required for DAT-1 export and function in vivo. We identified two conserved motifs ((584)KW(585) and (591)PYRKR(595)) where mutation results in a failure of GFP::DAT-1 to be efficiently exported to synapses and restore DAT-1 function. The (584)KW(585) motif conforms to a sequence proposed to support SEC24 binding, ER export from the endoplasmic reticulum (ER), and surface expression of mammalian DAT proteins, whereas the (591)PYRKR(595) sequence conforms to a 3R motif identified as a SEC24 binding site in vertebrate G-protein coupled receptors. Consistent with a potential role of SEC24 orthologs in DAT-1 export, we demonstrated DA neuron-specific expression of a sec-24.2 transcriptional reporter. Mutations of the orthologous C-terminal sequences in human DAT (hDAT) significantly reduced

  10. Analytical modelling of retrograde transport of nerve growth factors in an axon: a transient problem.

    PubMed

    Kuznetsov, A V

    2013-01-01

    The purpose of this paper was to develop an analytical solution describing retrograde transport of nerve growth factors (NGFs) from a target tissue to the neuron soma. The obtained solution is applied to describe two situations: (1) when all dynein motors are moving at a constant velocity and (2) when the dynein velocity distribution is described by a probability density function. The dynamics of NGF concentrations and fluxes is investigated. It is established that the magnitude of the diffusion flux forms a wave localised in space and propagating towards the neuron soma; the magnitude of this wave decreases as the wave propagates downstream. The dynein-driven flux of NGFs is demonstrated to be the main component in the NGF flux, which is shown to be strongly correlated with the NGF concentration.

  11. A temporal variation in nonneuronal protein synthesis in dorsal root ganglia and nerve and its significance to studies of axonal transport

    SciTech Connect

    Snyder, R.E.; O'Brien, D.W.; Nihei, T.

    1984-03-01

    Protein synthesis and fast axonal transport were studied in vitro using dorsal root ganglia (DRG)-sciatic nerve preparations from the amphibian Xenopus laevis. It was observed that the rate of incorporation of (/sup 3/H)leucine into protein in DRG and isolated segments of nerve began to increase 9 to 11 h after killing the animal, attaining at 13 to 17 h a maximum of 5- to 10-times preincrease (less than 9 h) values. At the same time as an increase in the rate of incorporation began, synthesis commenced in DRG and nerve exposed to cycloheximide (125 micrograms/ml). Whereas cycloheximide reduced fast axonal transport to 1 to 3% of control values in preparations maintained 20 to 24 h in vitro, cycloheximide reduced incorporation in DRG to only 80% of control values. N-terminal labeling studies showed that both the increased incorporation and cycloheximide-insensitive incorporation resulted from protein synthesis. Autoradiographic and incorporation studies indicated that nonneuronal cells situated in the ganglion capsule and perineural sheath of the nerve were responsible for both the increased incorporation and cycloheximide-insensitive synthesis. The findings have implications for the study of axonal transport.

  12. Age-dependent slowing of enteric axonal transport in insulin-resistant mice

    PubMed Central

    LePard, Kathy J; Cellini, Joseph

    2013-01-01

    AIM: To investigate retrograde tracer transport by gastric enteric neurons in insulin resistant mice with low or high glycosylated hemoglobin (Hb). METHODS: Under anesthesia, the retrograde tracer fluorogold was superficially injected into the fundus or antrum using a microsyringe in KK Cg-Ay/J mice prior to onset of type 2 diabetes mellitus (T2DM; 4 wk of age), at onset of T2DM (8 wk of age), and after 8, 16, or 24 wk of untreated T2DM and in age-matched KK/HIJ mice. Six days later, mice were sacrificed by CO2 narcosis followed by pneumothorax. Stomachs were removed and fixed. Sections from fundus, corpus and antrum were excised and mounted on a glass slide. Tracer-labeled neurons were viewed using a microscope and manually counted. Data were expressed as the number of neurons in short and long descending and ascending pathways and in local fundus and antrum pathways, and the number of neurons in all regions labeled after injection of tracer into either the fundus or the antrum. RESULTS: By 8 wk of age, body weights of KKAy mice (n = 12, 34 ± 1 g) were heavier than KK mice (n = 17, 29 ± 1 g; F (4, 120) = 4.414, P = 0.002] and glycosylated Hb was higher [KK: (n = 7), 4.97% ± 0.04%; KKAy: (n = 6), 6.57% ± 0.47%; F (1, 26) = 24.748, P < 0.001]. The number of tracer labeled enteric neurons was similar in KK and KKAy mice of all ages in the short descending pathway [F (1, 57) = 2.374, P = 0.129], long descending pathway [F (1, 57) = 0.922, P = 0.341], local fundus pathway [F (1, 53) = 2.464, P = 0.122], local antrum pathway [F (1, 57) = 0.728, P = 0.397], and short ascending pathway [F (1, 53) = 2.940, P = 0.092]. In the long ascending pathway, fewer tracer-labeled neurons were present in KKAy as compared to KK mice [KK: (n = 34), 302 ± 17; KKAy: (n = 29), 230 ± 15; F (1, 53) = 8.136, P = 0.006]. The number of tracer-labeled neurons was decreased in all mice by 16 wk as compared to 8 wk of age in the short descending pathway [8 wk: (n = 15), 305 ± 26; 16 wk: (n

  13. Varicella-zoster virus (VZV) infection of neurons derived from human embryonic stem cells: direct demonstration of axonal infection, transport of VZV, and productive neuronal infection.

    PubMed

    Markus, Amos; Grigoryan, Sergei; Sloutskin, Anna; Yee, Michael B; Zhu, Hua; Yang, In Hong; Thakor, Nitish V; Sarid, Ronit; Kinchington, Paul R; Goldstein, Ronald S

    2011-07-01

    Study of the human neurotrophic herpesvirus varicella-zoster virus (VZV) and of its ability to infect neurons has been severely limited by strict viral human tropism and limited availability of human neurons for experimentation. Human embryonic stem cells (hESC) can be differentiated to all the cell types of the body including neurons and are therefore a potentially unlimited source of human neurons to study their interactions with human neurotropic viruses. We report here reproducible infection of hESC-derived neurons by cell-associated green fluorescent protein (GFP)-expressing VZV. hESC-derived neurons expressed GFP within 2 days after incubation with mitotically inhibited MeWo cells infected with recombinant VZV expressing GFP as GFP fusions to VZV proteins or under an independent promoter. VZV infection was confirmed by immunostaining for immediate-early and viral capsid proteins. Infection of hESC-derived neurons was productive, resulting in release into the medium of infectious virions that appeared fully assembled when observed by electron microscopy. We also demonstrated, for the first time, VZV infection of axons and retrograde transport from axons to neuronal cell bodies using compartmented microfluidic chambers. The use of hESC-derived human neurons in conjunction with fluorescently tagged VZV shows great promise for the study of VZV neuronal infection and axonal transport and has potential for the establishment of a model for VZV latency in human neurons.

  14. Varicella-Zoster Virus (VZV) Infection of Neurons Derived from Human Embryonic Stem Cells: Direct Demonstration of Axonal Infection, Transport of VZV, and Productive Neuronal Infection▿

    PubMed Central

    Markus, Amos; Grigoryan, Sergei; Sloutskin, Anna; Yee, Michael B.; Zhu, Hua; Yang, In Hong; Thakor, Nitish V.; Sarid, Ronit; Kinchington, Paul R.; Goldstein, Ronald S.

    2011-01-01

    Study of the human neurotrophic herpesvirus varicella-zoster virus (VZV) and of its ability to infect neurons has been severely limited by strict viral human tropism and limited availability of human neurons for experimentation. Human embryonic stem cells (hESC) can be differentiated to all the cell types of the body including neurons and are therefore a potentially unlimited source of human neurons to study their interactions with human neurotropic viruses. We report here reproducible infection of hESC-derived neurons by cell-associated green fluorescent protein (GFP)-expressing VZV. hESC-derived neurons expressed GFP within 2 days after incubation with mitotically inhibited MeWo cells infected with recombinant VZV expressing GFP as GFP fusions to VZV proteins or under an independent promoter. VZV infection was confirmed by immunostaining for immediate-early and viral capsid proteins. Infection of hESC-derived neurons was productive, resulting in release into the medium of infectious virions that appeared fully assembled when observed by electron microscopy. We also demonstrated, for the first time, VZV infection of axons and retrograde transport from axons to neuronal cell bodies using compartmented microfluidic chambers. The use of hESC-derived human neurons in conjunction with fluorescently tagged VZV shows great promise for the study of VZV neuronal infection and axonal transport and has potential for the establishment of a model for VZV latency in human neurons. PMID:21525353

  15. Differential screening of mutated SOD1 transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons.

    PubMed

    Dupuis, L; de Tapia, M; René, F; Lutz-Bucher, B; Gordon, J W; Mercken, L; Pradier, L; Loeffler, J P

    2000-08-01

    In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.

  16. Biochemical analysis of axon-specific phosphorylation events using isolated squid axoplasms.

    PubMed

    Kang, Minsu; Baker, Lisa; Song, Yuyu; Brady, Scott T; Morfini, Gerardo

    2016-01-01

    Appropriate functionality of nodes of Ranvier, presynaptic terminals, and other axonal subdomains depends on efficient and timely delivery of proteins synthesized and packaged into membrane-bound organelles (MBOs) within the neuronal cell body. MBOs are transported and delivered to their final sites of utilization within axons by a cellular process known as fast axonal transport (FAT). Conventional kinesin, the most abundant multisubunit motor protein expressed in mature neurons, is responsible for FAT of a large variety of MBOs and plays a major role in the maintenance of appropriate axonal connectivity. Consistent with the variety and large number of discrete subdomains within axons, experimental evidence revealed the identity of several protein kinases that modulate specific functional activities of conventional kinesin. Thus, methods for the analysis of kinase activity and conventional kinesin phosphorylation facilitate the study of FAT regulation in health and disease conditions. Axonal degeneration, abnormal patterns of protein phosphorylation, and deficits in FAT represent early pathological features characteristic of neurological diseases caused by unrelated neuropathogenic proteins. Interestingly, some of these proteins were shown to produce deficits in FAT by modulating the activity of specific protein kinases involved in conventional kinesin phosphorylation. However, experimental systems that facilitate an evaluation of molecular events within axons remain scarce. Using the isolated squid axoplasm preparation, we describe methods for evaluating axon-autonomous effects of neuropathogenic proteins on the activity of protein kinases. Protocols are also provided to evaluate the effect of such proteins on the phosphorylation of endogenous axonal substrates, including conventional kinesin and neurofilaments.

  17. The axonal cytoskeleton: from organization to function

    PubMed Central

    Kevenaar, Josta T.; Hoogenraad, Casper C.

    2015-01-01

    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 structural basis for several specialized axonal structures, such as the axon initial segment (AIS) and presynaptic boutons. Emerging evidence suggest that the unique cytoskeleton organization in the axon is essential for its structure and integrity. In addition, the increasing number of neurodevelopmental and neurodegenerative diseases linked to defect in actin- and microtubule-dependent processes emphasizes the importance of a properly regulated cytoskeleton for normal axonal functioning. Here, we provide an overview of the current understanding of actin and microtubule organization within the axon and discuss models for the functional role of the cytoskeleton at specialized axonal structures. PMID:26321907

  18. The axonal cytoskeleton: from organization to function.

    PubMed

    Kevenaar, Josta T; Hoogenraad, Casper C

    2015-01-01

    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 structural basis for several specialized axonal structures, such as the axon initial segment (AIS) and presynaptic boutons. Emerging evidence suggest that the unique cytoskeleton organization in the axon is essential for its structure and integrity. In addition, the increasing number of neurodevelopmental and neurodegenerative diseases linked to defect in actin- and microtubule-dependent processes emphasizes the importance of a properly regulated cytoskeleton for normal axonal functioning. Here, we provide an overview of the current understanding of actin and microtubule organization within the axon and discuss models for the functional role of the cytoskeleton at specialized axonal structures.

  19. Flow characteristics in a crowded transport model

    NASA Astrophysics Data System (ADS)

    Burger, Martin; Pietschmann, Jan-Frederik

    2016-11-01

    The aim of this paper is to discuss the appropriate modelling of in- and outflow boundary conditions for nonlinear drift-diffusion models for the transport of particles including size exclusion and their effect on the behaviour of solutions. We use a derivation from a microscopic asymmetric exclusion process and its extension to particles entering or leaving on the boundaries. This leads to specific Robin-type boundary conditions for inflow and outflow, respectively. For the stationary equation we prove the existence of solutions in a suitable set-up. Moreover, we investigate the flow characteristics for a small diffusion parameter \\varepsilon , which yields the occurrence of a maximal current phase in addition to well-known one-sided boundary layer effects for linear drift-diffusion problems. In a 1D set-up we provide rigorous estimates in terms of ε, which confirm three different phases. Finally, we derive a numerical approach to solve the problem also in multiple dimensions.

  20. Mutant huntingtin's interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington's disease.

    PubMed

    Shirendeb, Ulziibat P; Calkins, Marcus J; Manczak, Maria; Anekonda, Vishwanath; Dufour, Brett; McBride, Jodi L; Mao, Peizhong; Reddy, P Hemachandra

    2012-01-15

    The purpose of this study was to investigate the link between mutant huntingtin (Htt) and neuronal damage in relation to mitochondria in Huntington's disease (HD). In an earlier study, we determined the relationship between mutant Htt and mitochondrial dynamics/synaptic viability in HD patients. We found mitochondrial loss, abnormal mitochondrial dynamics and mutant Htt association with mitochondria in HD patients. In the current study, we sought to expand on our previous findings and further elucidate the relationship between mutant Htt and mitochondrial and synaptic deficiencies. We hypothesized that mutant Htt, in association with mitochondria, alters mitochondrial dynamics, leading to mitochondrial fragmentation and defective axonal transport of mitochondria in HD neurons. In this study, using postmortem HD brains and primary neurons from transgenic BACHD mice, we identified mutant Htt interaction with the mitochondrial protein Drp1 and factors that cause abnormal mitochondrial dynamics, including GTPase Drp1 enzymatic activity. Further, using primary neurons from BACHD mice, for the first time, we studied axonal transport of mitochondria and synaptic degeneration. We also investigated the effect of mutant Htt aggregates and oligomers in synaptic and mitochondrial deficiencies in postmortem HD brains and primary neurons from BACHD mice. We found that mutant Htt interacts with Drp1, elevates GTPase Drp1 enzymatic activity, increases abnormal mitochondrial dynamics and results in defective anterograde mitochondrial movement and synaptic deficiencies. These observations support our hypothesis and provide data that can be utilized to develop therapeutic targets that are capable of inhibiting mutant Htt interaction with Drp1, decreasing mitochondrial fragmentation, enhancing axonal transport of mitochondria and protecting synapses from toxic insults caused by mutant Htt.

  1. A New Regulatory Mechanism for Kv7.2 Protein During Neuropathy: Enhanced Transport from the Soma to Axonal Terminals of Injured Sensory Neurons

    PubMed Central

    Cisneros, Elsa; Roza, Carolina; Jackson, Nieka; López-García, José Antonio

    2015-01-01

    Kv7.2 channel expression has been reported to decrease in dorsal root ganglia (DRG) following the induction of a peripheral neuropathy while other experiments show that Kv7.2 accumulates in peripheral neuromas. The mechanisms underlying these novel expression patterns are poorly understood. Here we use immunofluorescence methods to analyze Kv7.2 protein expression changes in sensory neurons following peripheral axotomy and the potential role of axonal transport. Results indicate that DRG neurons express Kv7.2 in ~16% of neurons and that this number decreases by about 65% after axotomy. Damaged neurons were identified in DRG by application of the tracer Fluoro-ruby at the site of injury during surgery. Reduction of Kv7.2 expression was particularly strong in damaged neurons although some loss was also found in putative uninjured neurons. In parallel to the decrease in the soma of axotomized sensory neurons, Kv7.2 accumulated at neuromatose fiber endings. Blockade of axonal transport with either vinblastine (VLB) or colchicine (COL) abolished Kv7.2 redistribution in neuropathic animals. Channel distribution rearrangements did not occur following induction of inflammation in the hind paw. Behavioral tests indicate that protein rearrangements within sensory afferents are essential to the development of allodynia under neuropathic conditions. These results suggest that axotomy enhances axonal transport in injured sensory neurons, leading to a decrease of somatic expression of Kv7.2 protein and a concomitant accumulation in damaged fiber endings. Localized changes in channel expression patterns under pathological conditions may create novel opportunities for Kv7.2 channel openers to act as analgesics. PMID:26696829

  2. Light and electron microscopic analysis of enkephalin-like immunoreactivity in the basolateral amygdala, including evidence for convergence of enkephalin-containing axon terminals and norepinephrine transporter-containing axon terminals onto common targets

    PubMed Central

    Zhang, Jingyi; McDonald, Alexander J.

    2016-01-01

    Modulatory interactions of opioids and norepinephrine (NE) in the anterior subdivision of the basolateral nucleus of the amygdala (BLa) are critical for the consolidation of memories of emotionally arousing experiences. Although there have been several studies of the noradrenergic system in the amygdalar basolateral nuclear complex (BLC), little is known about the chemical neuroanatomy of opioid systems in this region. To address this knowledge gap the present study first examined the distribution of met-enkephalin-like immunoreactivity (ENK-ir) in the BLC at the light microscopic level, and then utilized dual-labeling immunocytochemistry combined with electron microscopy to investigate the extent of convergence of NE and ENK terminals onto common structures in the BLa. Antibodies to ENK and the norepinephrine transporter (NET) were used in these studies. Light microscopic examination revealed that a subpopulation of small nonpyramidal neurons expressed ENK-ir in all nuclei of the BLC. In addition, the somata of some pyramidal cells exhibited light to moderate ENK-ir. ENK+ axon terminals were also observed. Ultrastructural analysis confined to the BLa revealed that most ENK+ axon terminals formed asymmetrical synapses that mainly contacted spines and shafts of thin dendrites. ENK+ terminals forming symmetrical synapses mainly contacted dendritic shafts. Approximately 20% of NET+ terminals contacted a structure that was also contacted by an ENK+ terminal and 6% of NET+ terminals contacted an ENK+ terminal. These findings suggest that ENK and NE terminals in the BLa may interact by targeting common dendrites and by direct interactions between the two types of terminals. PMID:26835559

  3. Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity

    SciTech Connect

    Zarbin, M.A.; Wamsley, J.K.; Kuhar, M.J.

    1982-07-01

    The presence and transport of muscarinic cholinergic binding sites have been detected in the rat vagus nerve. These binding sites accumulate both proximal and distal to ligatures in a time-dependent manner. The results of double ligature and colchicine experiments are compatible with the notion that the anterogradely transported binding sites move by fast transport. Most of the sites accumulating proximal to ligatures bind the agonist carbachol with high affinity, while most of the sites accumulating distally bind carbachol with a low affinity. Also, the receptors transported in the anterograde direction are affected by a guanine nucleotide analogue (GppNHp), while those transported in the retrograde direction are less, or not, affected. The bulk of the sites along the unligated nerve trunk bind carbachol with a low affinity and are less sensitive to GppNHp modulation than the anterogradely transported sites. These results suggest that some receptors in the vagus may undergo axonal transport in association with regulatory proteins and that receptor molecules undergo changes in their binding and regulatory properties during their life cycle. These data also support the notion that the high and low affinity agonist form of the muscarinic receptor represent different modulated forms of a single receptor molecule.

  4. UNC-16 (JIP3) Acts Through Synapse-Assembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons.

    PubMed

    Edwards, Stacey L; Morrison, Logan M; Yorks, Rosalina M; Hoover, Christopher M; Boominathan, Soorajnath; Miller, Kenneth G

    2015-09-01

    The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16's organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-α). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(-) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADα (a SAD-1-interacting protein) in the CSS system. In an unc-16(-) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(-) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16's organelle transport regulatory function. Copyright © 2015 by the Genetics Society of America.

  5. Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts Axonal Transport in a Drosophila HSP Model

    PubMed Central

    Stanchev, Doychin T.; Schneider, Carola D.; Karle, Kathrin N.; Daub, Katharina J.; Siegert, Vera K.; Flötenmeyer, Matthias; Schwarz, Heinz; Schöls, Ludger; Rasse, Tobias M.

    2012-01-01

    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. PMID:23209432

  6. Axon specification in hippocampal neurons.

    PubMed

    Fukata, Yuko; Kimura, Toshihide; Kaibuchi, Kozo

    2002-08-01

    Neurons are the most highly polarized cells, comprised of two structurally and functionally distinct parts, axons and dendrites. This asymmetry enables a vectorial flow of signaling within neurons. One of the most fundamental questions still to be answered in neuroscience is how these two specialized processes initially develop. The first manifestation of polarization occurs when one of the immature neurites acquires axonal characteristics. We review recent advances that have highlighted the involvement of several cellular events in the initial formation of the axon, including membrane traffic and cytoskeletal rearrangement. We then discuss the molecular mechanisms underlying axon formation, focusing on the Rho family small GTPases and an axon-inducing neuronal protein, CRMP-2.

  7. Evaluation of Retinal Nerve Fiber Layer Thickness and Axonal Transport 1 and 2 Weeks After 8 Hours of Acute Intraocular Pressure Elevation in Rats

    PubMed Central

    Abbott, Carla J.; Choe, Tiffany E.; Lusardi, Theresa A.; Burgoyne, Claude F.; Wang, Lin; Fortune, Brad

    2014-01-01

    Purpose. To compare in vivo retinal nerve fiber layer thickness (RNFLT) and axonal transport at 1 and 2 weeks after an 8-hour acute IOP elevation in rats. Methods. Forty-seven adult male Brown Norway rats were used. Procedures were performed under anesthesia. The IOP was manometrically elevated to 50 mm Hg or held at 15 mm Hg (sham) for 8 hours unilaterally. The RNFLT was measured by spectral-domain optical coherence tomography. Anterograde and retrograde axonal transport was assessed from confocal scanning laser ophthalmoscopy imaging 24 hours after bilateral injections of 2 μL 1% cholera toxin B-subunit conjugated to AlexaFluor 488 into the vitreous or superior colliculi, respectively. Retinal ganglion cell (RGC) and microglial densities were determined using antibodies against Brn3a and Iba-1. Results. The RNFLT in experimental eyes increased from baseline by 11% at 1 day (P < 0.001), peaked at 19% at 1 week (P < 0.0001), remained 11% thicker at 2 weeks (P < 0.001), recovered at 3 weeks (P > 0.05), and showed no sign of thinning at 6 weeks (P > 0.05). There was no disruption of anterograde transport at 1 week (superior colliculi fluorescence intensity, 75.3 ± 7.9 arbitrary units [AU] for the experimental eyes and 77.1 ± 6.7 AU for the control eyes) (P = 0.438) or 2 weeks (P = 0.188). There was no obstruction of retrograde transport at 1 week (RCG density, 1651 ± 153 per mm2 for the experimental eyes and 1615 ± 135 per mm2 for the control eyes) (P = 0.63) or 2 weeks (P = 0.25). There was no loss of Brn3a-positive RGC density at 6 weeks (P = 0.74) and no increase in microglial density (P = 0.92). Conclusions. Acute IOP elevation to 50 mm Hg for 8 hours does not cause a persisting axonal transport deficit at 1 or 2 weeks or a detectable RNFLT or RGC loss by 6 weeks but does lead to transient RNFL thickening that resolves by 3 weeks. PMID:24398096

  8. Transport characteristics across drum filter vents and polymer bags

    SciTech Connect

    Liekhus, K.J.

    1994-08-01

    The rate at which hydrogen (H {sub 2}) or a volatile organic compound (VOC) exits a layer of confinement in a vented waste drum is proportional to the concentration difference across the layer. The proportionality constant is the gas transport characteristic. A series of transport experiments were conducted to determine H{sub 2} and VOC transport characteristics across different drum filter vents and polymer bags. This report reviews the methods and results of past investigators in defining transport characteristics across filter vents and polymer bags, describes the apparatus and procedures used in these experiments, compares the reported and estimated transport characteristics with earlier results, and discusses the impact of changing the transport characteristic values used in model calculations.

  9. Axonal transport of TDP-43 mRNA granules in neurons is impaired by ALS-causing mutations

    PubMed Central

    Carrasco, Monica A.; Williams, Luis A.; Winborn, Christina S.; Han, Steve S. W.; Kiskinis, Evangelos; Winborn, Brett; Freibaum, Brian D.; Kanagaraj, Anderson; Clare, Alison J.; Badders, Nisha M.; Bilican, Bilada; Chaum, Edward; Chandran, Siddharthan; Shaw, Christopher E.; Eggan, Kevin C.; Maniatis, Tom; Taylor, J. Paul

    2014-01-01

    Summary The RNA binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43. PMID:24507191

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

    PubMed

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

    2015-11-01

    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.

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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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. PMID:26398944

  12. Upregulation and axonal transport of synaptotagmin-IV in the direct-pathway medium spiny neurons in hemi-parkinsonian rats induced by dopamine D1 receptor stimulation.

    PubMed

    Tratnjek, Larisa; Glavan, Gordana; Višnjar, Tanja; Živin, Marko

    2016-04-01

    Synaptotagmin-IV (Syt-IV) may function as a regulator of Ca(2+) -dependent synaptic transmission. In the hemi-parkinsonian rats with unilateral lesions of dopaminergic nigrostriatal neurons Syt-IV and substance-P (SP) mRNAs could be upregulated within the dopaminergically hypersensitive striatum of the lesioned brain hemisphere via the stimulation of striatal dopamine D1 (D1-R), but not D2 receptors. The hypersensitive D1-R-mediated transmission may be the culprit for the undesired expression of levodopa-induced dyskinesia, implying the involvement of Syt-IV and SP in the process. First, striatal cellular phenotypes expressing Syt-IV were determined. It was found to be expressed in all striatal neurons and a small population of astrocytes. Then it was examined, if the D1-R-mediated upregulation of Syt-IV mRNA may result in the upregulation of the translated protein. It was found that, after acute stimulation with a selective D1 agonist, (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-82958), Syt-IV was elevated within the SP-expressing striatal neurons of the lesioned side. This was followed by the upregulation of Syt-IV, but not of its mRNA, within the ipsilateral target nuclei of the direct-pathway medium spiny neurons, indicating axonal transport of de novo synthesized protein to their SP-positive synaptic terminals. However, despite the striatal upregulation of SP and Syt-IV following a similar time-course, their subcellular co-localization within the axonal terminals was not found. It was therefore suggested that Syt-IV may regulate the hypersensitive striatal synaptic transmission, although via a SP-independent mechanism. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  13. Cell intrinsic control of axon regeneration

    PubMed Central

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

    2014-01-01

    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

  14. Simulating tubulin-associated unit transport in an axon: using bootstrapping for estimating confidence intervals of best-fit parameter values obtained from indirect experimental data.

    PubMed

    Kuznetsov, I A; Kuznetsov, A V

    2017-05-01

    In this paper, we first develop a model of axonal transport of tubulin-associated unit (tau) protein. We determine the minimum number of parameters necessary to reproduce published experimental results, reducing the number of parameters from 18 in the full model to eight in the simplified model. We then address the following questions: Is it possible to estimate parameter values for this model using the very limited amount of published experimental data? Furthermore, is it possible to estimate confidence intervals for the determined parameters? The idea that is explored in this paper is based on using bootstrapping. Model parameters were estimated by minimizing the objective function that simulates the discrepancy between the model predictions and experimental data. Residuals were then identified by calculating the differences between the experimental data and model predictions. New, surrogate 'experimental' data were generated by randomly resampling residuals. By finding sets of best-fit parameters for a large number of surrogate data the histograms for the model parameters were produced. These histograms were then used to estimate confidence intervals for the model parameters, by using the percentile bootstrap. Once the model was calibrated, we applied it to analysing some features of tau transport that are not accessible to current experimental techniques.

  15. High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport.

    PubMed

    Yi, Julie Y; Ori-McKenney, Kassandra M; McKenney, Richard J; Vershinin, Michael; Gross, Steven P; Vallee, Richard B

    2011-10-17

    The specific physiological roles of dynein regulatory factors remain poorly understood as a result of their functional complexity and the interdependence of dynein and kinesin motor activities. We used a novel approach to overcome these challenges, combining acute in vivo inhibition with automated high temporal and spatial resolution particle tracking. Acute dynein inhibition in nonneuronal cells caused an immediate dispersal of diverse forms of cargo, resulting from a sharp decrease in microtubule minus-end run length followed by a gradual decrease in plus-end runs. Acute LIS1 inhibition or LIS1 RNA interference had little effect on lysosomes/late endosomes but severely inhibited axonal transport of large, but not small, vesicular structures. Our acute inhibition results argue against direct mechanical activation of opposite-directed motors and offer a novel approach of potential broad utility in the study of motor protein function in vivo. Our data also reveal a specific but cell type-restricted role for LIS1 in large vesicular transport and provide the first quantitative support for a general role for LIS1 in high-load dynein functions.

  16. Stratigraphic control of flow and transport characteristics.

    PubMed

    Edington, Dwaine; Poeter, Eileen

    2007-01-01

    Ground water flow and travel time are dependent on stratigraphic architecture, which is governed by competing processes that control the spatial and temporal distribution of accommodation and sediment supply. Accommodation is the amount of space in which sediment may accumulate as defined by the difference between the energy gradient and the topographic surface. The temporal and spatial distribution of accommodation is affected by processes that change the distribution of energy (e.g., sea level or subsidence). Fluvial stratigraphic units, generated by FLUVSIM (a stratigraphic simulator based on accommodation and sediment supply), with varying magnitudes and causes of accommodation, were incorporated into a hydraulic regime using MODFLOW (a ground water flow simulator), and particles were tracked using MODPATH (a particle-tracking algorithm). These experiments illustrate that the dominant type of accommodation process influences the degree of continuity of stratigraphic units and thus affects ground water flow and transport. When the hydraulic gradient is parallel to the axis of the fluvial system in the depositional environment, shorter travel times occur in low-total accommodation environments and longer travel times in high-total accommodation environments. Given the same total accommodation, travel times are longer when sea-level change is the dominant process than those in systems dominated by subsidence.

  17. Cortical Divergent Projections in Mice Originate from Two Sequentially Generated, Distinct Populations of Excitatory Cortical Neurons with Different Initial Axonal Outgrowth Characteristics.

    PubMed

    Hatanaka, Yumiko; Namikawa, Tomohiro; Yamauchi, Kenta; Kawaguchi, Yasuo

    2016-05-01

    Excitatory cortical neurons project to various subcortical and intracortical regions, and exhibit diversity in their axonal connections. Although this diversity may develop from primary axons, how many types of axons initially occur remains unknown. Using a sparse-labeling in utero electroporation method, we investigated the axonal outgrowth of these neurons in mice and correlated the data with axonal projections in adults. Examination of lateral cortex neurons labeled during the main period of cortical neurogenesis (E11.5-E15.5) indicated that axonal outgrowth commonly occurs in the intermediate zone. Conversely, the axonal direction varied; neurons labeled before E12.5 and the earliest cortical plate neurons labeled at E12.5 projected laterally, whereas neurons labeled thereafter projected medially. The expression of Ctip2 and Satb2 and the layer destinations of these neurons support the view that lateral and medial projection neurons are groups of prospective subcortical and callosal projection neurons, respectively. Consistently, birthdating experiments demonstrated that presumptive lateral projection neurons were generated earlier than medial projection neurons, even within the same layer. These results suggest that the divergent axonal connections of excitatory cortical neurons begin from two types of primary axons, which originate from two sequentially generated distinct subpopulations: early-born lateral (subcortical) and later-born medial (callosal) projection neuron groups.

  18. The developmentally regulated expression of Menkes protein ATP7A suggests a role in axon extension and synaptogenesis.

    PubMed

    El Meskini, Rajaâ; Cline, Laura B; Eipper, Betty A; Ronnett, Gabriele V

    2005-01-01

    Menkes disease (MD) is a neurodegenerative disorder caused by mutation of the copper transporter ATP7A. While several enzymes expressed in mature neurons require copper, MD neurodegenerative changes cannot be explained by known requirements for ATP7A in neuronal development. To investigate additional roles for ATP7A during development, we characterized its pattern of expression using the olfactory system as a neurodevelopmental model. ATP7A expression in neurons was developmentally regulated rather than constitutively. Initially expressed in the cell bodies of developing neurons, ATP7A protein later shifted to extending axons, peaking prior to synaptogenesis. Similarly, after injury-stimulated neurogenesis, ATP7A expression increased in neurons and axons preceding synaptogenesis. Interestingly, copper-transport-deficient ATP7A still exhibits axonal localization. These results support a role for ATP7A in axon extension, which may contribute to the severe neurodegeneration characteristic of MD.

  19. Retinal projections in the short-tailed fruit bat, Carollia perspicillata, as studied using the axonal transport of cholera toxin B subunit: Comparison with mouse.

    PubMed

    Scalia, Frank; Rasweiler, John J; Danias, John

    2015-08-15

    To provide a modern description of the Chiropteran visual system, the subcortical retinal projections were studied in the short-tailed fruit bat, Carollia perspicillata, using the anterograde transport of eye-injected cholera toxin B subunit, supplemented by the silver-impregnation of anterograde degeneration following eye removal, and compared with the retinal projections of the mouse. The retinal projections were heavily labeled by the transported toxin in both species. Almost all components of the murine retinal projection are present in Carollia in varying degrees of prominence and laterality. The projections: to the superior colliculus, accessory optic nuclei, and nucleus of the optic tract are predominantly or exclusively contralateral; to the dorsal lateral geniculate nucleus and posterior pretectal nucleus are predominantly contralateral; to the ventral lateral geniculate nucleus, intergeniculate leaflet, and olivary pretectal nucleus have a substantial ipsilateral component; and to the suprachiasmatic nucleus are symmetrically bilateral. The retinal projection in Carollia is surprisingly reduced at the anterior end of the dorsal lateral geniculate and superior colliculus, suggestive of a paucity of the relevant ganglion cells in the ventrotemporal retina. In the superior colliculus, in which the superficial gray layer is very thin, the projection is patchy in places where the layer is locally absent. Except for a posteriorly located lateral terminal nucleus, the other accessory optic nuclei are diminutive in Carollia, as is the nucleus of the optic tract. In both species the cholera toxin labeled sparse groups of apparently terminating axons in numerous regions not listed above. A question of their significance is discussed.

  20. Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration in a mouse model of Alzheimer's disease.

    PubMed

    Calkins, Marcus J; Manczak, Maria; Mao, Peizhong; Shirendeb, Ulziibat; Reddy, P Hemachandra

    2011-12-01

    Increasing evidence suggests that the accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria causes synaptic mitochondrial failure and synaptic degeneration in Alzheimer's disease (AD). The purpose of this study was to better understand the effects of Aβ in mitochondrial activity and synaptic alterations in neurons from a mouse model of AD. Using primary neurons from a well-characterized Aβ precursor protein transgenic (AβPP) mouse model (Tg2576 mouse line), for the first time, we studied mitochondrial activity, including axonal transport of mitochondria, mitochondrial dynamics, morphology and function. Further, we also studied the nature of Aβ-induced synaptic alterations, and cell death in primary neurons from Tg2576 mice, and we sought to determine whether the mitochondria-targeted antioxidant SS31 could mitigate the effects of oligomeric Aβ. We found significantly decreased anterograde mitochondrial movement, increased mitochondrial fission and decreased fusion, abnormal mitochondrial and synaptic proteins and defective mitochondrial function in primary neurons from AβPP mice compared with wild-type (WT) neurons. Transmission electron microscopy revealed a large number of small mitochondria and structurally damaged mitochondria, with broken cristae in AβPP primary neurons. We also found an increased accumulation of oligomeric Aβ and increased apoptotic neuronal death in the primary neurons from the AβPP mice relative to the WT neurons. Our results revealed an accumulation of intraneuronal oligomeric Aβ, leading to mitochondrial and synaptic deficiencies, and ultimately causing neurodegeneration in AβPP cultures. However, we found that the mitochondria-targeted antioxidant SS31 restored mitochondrial transport and synaptic viability, and decreased the percentage of defective mitochondria, indicating that SS31 protects mitochondria and synapses from Aβ toxicity.

  1. Transport synthetic acceleration for long-characteristics assembly-level transport problems

    SciTech Connect

    Zika, M.R.; Adams, M.L.

    2000-02-01

    The authors apply the transport synthetic acceleration (TSA) scheme to the long-characteristics spatial discretization for the two-dimensional assembly-level transport problem. This synthetic method employs a simplified transport operator as its low-order approximation. Thus, in the acceleration step, the authors take advantage of features of the long-characteristics discretization that make it particularly well suited to assembly-level transport problems. The main contribution is to address difficulties unique to the long-characteristics discretization and produce a computationally efficient acceleration scheme. The combination of the long-characteristics discretization, opposing reflecting boundary conditions (which are present in assembly-level transport problems), and TSA presents several challenges. The authors devise methods for overcoming each of them in a computationally efficient way. Since the boundary angular data exist on different grids in the high- and low-order problems, they define restriction and prolongation operations specific to the method of long characteristics to map between the two grids. They implement the conjugate gradient (CG) method in the presence of opposing reflection boundary conditions to solve the TSA low-order equations. The CG iteration may be applied only to symmetric positive definite (SPD) matrices; they prove that the long-characteristics discretization yields an SPD matrix. They present results of the acceleration scheme on a simple test problem, a typical pressurized water reactor assembly, and a typical boiling water reactor assembly.

  2. Superballistic characteristics in transient phonon ballistic-diffusive transport

    NASA Astrophysics Data System (ADS)

    Tang, Dao-Sheng; Cao, Bing-Yang

    2017-09-01

    While diffusive, superdiffusive, and ballistic phonon transports have been widely investigated, the superballistic phenomenon, where the time index of the energy mean square displacement with respect to time is greater than 2, has been neither predicted nor observed. In this work, we report on the superballistic characteristics obtained from simulations of transient phonon ballistic-diffusive transport both during and after the input of a heat pulse into a nanoscale film. The superballistic behaviors are well described by a previously proposed model for electron wave packet spreading employing a point source and further explained by the superposition effect of heat pulses. The relative superposition time, a dimensionless parameter, is defined to describe the degree of the heat pulse superposition. The analysis of superballistic characteristics in this work is expected to guide experiments for detecting the phonon superballistic transport. Also, it provides a potential phenomenological description for the superballistic phenomena in more complex systems.

  3. Neutron transport in WIMS by the characteristics method

    SciTech Connect

    Halsall, M.J. )

    1993-01-01

    The common methods of solving the neutron transport equation in reactor assembly geometries involve some geometric approximation. The standard differential transport methods and diffusion methods rely on pin-cell smearing, and transmission probability methods make approximations to the boundary fluxes linking pin cells. Integral transport methods (collision probabilities) can cope with pin geometries by numerical integration but require excessive computing times that increase with the square of the number of regions. The characteristics method in WIMS, known as CACTUS, solves the differential transport equation by a numerical tracking technique whose accuracy is limited only by computing resources; in its WIMS implementation it can handle any pin-type geometry without the need for preliminary spatial smearing.

  4. Torsional Behavior of Axonal Microtubule Bundles

    PubMed Central

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

    2015-01-01

    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

  5. TRANSPORT CHARACTERISTICS OF SELECTED PWR LOCA GENERATED DEBRIS.

    SciTech Connect

    A. K. MAJI; B. MARSHALL; ET AL

    2000-10-01

    In the unlikely event of a Loss of Coolant Accident (LOCA) in a pressurized water reactor (PWR), break jet impingement would dislodge thermal insulation from nearby piping, as well as other materials within the containment, such as paint chips, concrete dust, and fire barrier materials. Steam/water flows induced by the break and by the containment sprays would transport debris to the containment floor. Subsequently, debris would likely transport to and accumulate on the suction sump screens of the emergency core cooling system (ECCS) pumps, thereby potentially degrading ECCS performance and possibly even failing the ECCS. In 1998, the U. S. Nuclear Regulatory Commission (NRC) initiated a generic study (Generic Safety Issue-191) to evaluate the potential for the accumulation of LOCA related debris on the PWR sump screen and the consequent loss of ECCS pump net positive suction head (NPSH). Los Alamos National Laboratory (LANL), supporting the resolution of GSI-191, was tasked with developing a method for estimating debris transport in PWR containments to estimate the quantity of debris that would accumulate on the sump screen for use in plant specific evaluations. The analytical method proposed by LANL, to predict debris transport within the water that would accumulate on the containment floor, is to use computational fluid dynamics (CFD) combined with experimental debris transport data to predict debris transport and accumulation on the screen. CFD simulations of actual plant containment designs would provide flow data for a postulated accident in that plant, e.g., three-dimensional patterns of flow velocities and flow turbulence. Small-scale experiments would determine parameters defining the debris transport characteristics for each type of debris. The containment floor transport methodology will merge debris transport characteristics with CFD results to provide a reasonable and conservative estimate of debris transport within the containment floor pool and

  6. Side-To-Side Nerve Bridges Support Donor Axon Regeneration Into Chronically Denervated Nerves and Are Associated With Characteristic Changes in Schwann Cell Phenotype.

    PubMed

    Hendry, J Michael; Alvarez-Veronesi, M Cecilia; Snyder-Warwick, Alison; Gordon, Tessa; Borschel, Gregory H

    2015-11-01

    Chronic denervation resulting from long nerve regeneration times and distances contributes greatly to suboptimal outcomes following nerve injuries. Recent studies showed that multiple nerve grafts inserted between an intact donor nerve and a denervated distal recipient nerve stump (termed "side-to-side nerve bridges") enhanced regeneration after delayed nerve repair. To examine the cellular aspects of axon growth across these bridges to explore the "protective" mechanism of donor axons on chronically denervated Schwann cells. In Sprague Dawley rats, 3 side-to-side nerve bridges were placed over a 10-mm distance between an intact donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) distal nerve stump. Green fluorescent protein-expressing TIB axons grew across the bridges and were counted in cross section after 4 weeks. Immunofluorescent axons and Schwann cells were imaged over a 4-month period. Denervated Schwann cells dedifferentiated to a proliferative, nonmyelinating phenotype within the bridges and the recipient denervated CP nerve stump. As donor TIB axons grew across the 3 side-to-side nerve bridges and into the denervated CP nerve, the Schwann cells redifferentiated to the myelinating phenotype. Bridge placement led to an increased mass of hind limb anterior compartment muscles after 4 months of denervation compared with muscles whose CP nerve was not "protected" by bridges. This study describes patterns of donor axon regeneration and myelination in the denervated recipient nerve stump and supports a mechanism where these donor axons sustain a proregenerative state to prevent deterioration in the face of chronic denervation.

  7. The formation of axonal caliber and nodes of Ranvier

    NASA Astrophysics Data System (ADS)

    Li, Yinyun; Jung, Peter; Brown, Anthony

    2013-03-01

    A remarkable feature of myelinated neurons is that their axons are constricted at the nodes of Ranvier. These are the locations where axons are directly exposed to the extracellular space and where the vast majority of the ion channels are located. These constrictions emerge during development and have been observed to reduce axonal cross sectional area by factors of more than 10. Combining fluorescent imaging methods with computational modeling, we describe how the nervous system regulates the local caliber of its axons through the regulation of the transport kinetics of its most important cytoskeletal elements, the neurofilaments, matching axon caliber and shape to its physiologic function. National Science Foundation IOS 1146789

  8. Voltage-sensitive dye recording using retrogradely transported dye in the chicken spinal cord: staining and signal characteristics.

    PubMed

    Wenner, P; Tsau, Y; Cohen, L B; O'Donovan, M J; Dan, Y

    1996-12-28

    We describe a novel method for retrogradely labeling specific neuronal populations using voltage-sensitive dyes. Styryl dyes were injected into the ventral roots of the isolated embryonic chick spinal cord. After waiting several hours, the dye labeled motoneurons and autonomic preganglionic neurons. Neuronal cell bodies, dendrites and axons were labeled; we presume that the dye traveled either by retrograde transport or by diffusion within the membrane of the axon to which the dyes were initially applied. Using either a photodiode array or a photomultiplier, fluorescence changes could be recorded from motoneurons following antidromic or synaptic activation. Several characteristics of the fluorescence changes were measured indicating that the signals did indeed reflect changes in the motoneuron membrane potential. The best labeling and optical signals were obtained using the relatively hydrophobic dyes di-8-ANEPPQ and di-12-ANEPEQ. In the great majority of cases these dyes responded with an increase in fluorescence of 1-3% (delta F/F) in response to synaptic or antidromic depolarization of the motoneurons. We anticipate that these techniques should be useful in the mapping of activity patterns and connectivity in neural networks within a defined population of neurons.

  9. Axonal degeneration and axonal caliber alterations following combined beta,beta'-iminodipropionitrile (IDPN) and acrylamide administration.

    PubMed

    Gold, B G; Halleck, M M

    1989-11-01

    A new model of neurofilamentous axonal abnormality is described which employs combined administration of beta,beta'-iminodipropionitrile (IDPN) and acrylamide (AC). The model was developed to test the hypothesis that IDPN-induced swelling increases the vulnerability of the distal axon to a second neurotoxic chemical insult. Rats were given a single intraperitoneal (IP) injection of IDPN (1.5 g/kg) one week before receiving a single injection of AC (75 mg/kg, IP). Axonal degeneration was observed at multiple levels along the sciatic nerve at two weeks (with reference to IDPN administration), and was not progressive up to five weeks. Quantitation of degenerating fibers demonstrated that the extent of degeneration increased distally along the sciatic nerve. Single administration of either IDPN or AC did not produce degeneration. Thus, IDPN-induced neurofilamentous swellings alter the susceptibility of the axon to AC neurotoxicity. Two variations of this model were also studied. First, rats given five daily injections of AC (30 mg/kg, IP) beginning one week following IDPN administration developed accumulations of fast axonally transported materials in IDPN-induced microtubule channels. Second, rats given chronic injections of AC (30 mg/kg, IP, five days/week, for four weeks), to reduce the delivery of neurofilaments to the proximal axon, developed less prominent axonal enlargements when challenged with IDPN. Thus, axonal atrophy can mask the development of neurofilamentous axonal swellings.

  10. Optofluidic control of axonal guidance

    NASA Astrophysics Data System (ADS)

    Gu, Ling; Ordonez, Simon; Black, Bryan; Mohanty, Samarendra K.

    2013-03-01

    Significant efforts are being made for control on axonal guidance due to its importance in nerve regeneration and in the formation of functional neuronal circuitry in-vitro. These include several physical (topographic modification, optical force, and electric field), chemical (surface functionalization cues) and hybrid (electro-chemical, photochemical etc) methods. Here, we report comparison of the effect of linear flow versus microfluidic flow produced by an opticallydriven micromotor in guiding retinal ganglion axons. A circularly polarized laser tweezers was used to hold, position and spin birefringent calcite particle near growth cone, which in turn resulted in microfluidic flow. The flow rate and resulting shear-force on axons could be controlled by a varying the power of the laser tweezers beam. The calcite particles were placed separately in one chamber and single particle was transported through microfluidic channel to another chamber containing the retina explant. In presence of flow, the turning of axons was found to strongly correlate with the direction of flow. Turning angle as high as 90° was achieved. Optofluidic-manipulation can be applied to other types of mammalian neurons and also can be extended to stimulate mechano-sensing neurons.

  11. Empirical relations between large wood transport and catchment characteristics

    NASA Astrophysics Data System (ADS)

    Steeb, Nicolas; Rickenmann, Dieter; Rickli, Christian; Badoux, Alexandre

    2017-04-01

    The transport of vast amounts of large wood (LW) in water courses can considerably aggravate hazardous situations during flood events, and often strongly affects resulting flood damage. Large wood recruitment and transport are controlled by various factors which are difficult to assess and the prediction of transported LW volumes is difficult. Such information are, however, important for engineers and river managers to adequately dimension retention structures or to identify critical stream cross-sections. In this context, empirical formulas have been developed to estimate the volume of transported LW during a flood event (Rickenmann, 1997; Steeb et al., 2017). The data base of existing empirical wood load equations is, however, limited. The objective of the present study is to test and refine existing empirical equations, and to derive new relationships to reveal trends in wood loading. Data have been collected for flood events with LW occurrence in Swiss catchments of various sizes. This extended data set allows us to derive statistically more significant results. LW volumes were found to be related to catchment and transport characteristics, such as catchment size, forested area, forested stream length, water discharge, sediment load, or Melton ratio. Both the potential wood load and the fraction that is effectively mobilized during a flood event (effective wood load) are estimated. The difference of potential and effective wood load allows us to derive typical reduction coefficients that can be used to refine spatially explicit GIS models for potential LW recruitment.

  12. Relativistic Electron Beam Transport and Characteristics in Solid Density Plasmas

    SciTech Connect

    Snavely, R A; King, J; Freeman, R R; Hatchett, S; Key, M H; Koch, J; Langdon, A B; Lasinsky, B; MacKinnon, A; Wilks, S; Stephens, R

    2003-08-13

    The transport of intense relativistic beams in solid density plasma presently is actively being studied in laser laboratories around the world. The correct understanding of the transport enables further application of fast laser driven electrons to a host of interesting uses. Advanced x-ray sources, proton and ion beam generation and plasma heating in fast ignitor fusion all are owed their eventual utility to this transport. We report on measurements of relativistic transport over the whole of the transport region, via analysis of x-ray emission. Our experiments cover laser powers from Terawatt to Petawatt. Advances in transverse imaging of fluorescent k-alpha x-rays generated along the electron beam path are used to diagnose the electron emission. Additionally the spatial pattern of Bremsstrahlung x-rays provides clues into the physics of electron transport in above Alfven current limit beams. Issues regarding the electron distribution function will be discussed in light of possible electron transport anomalies. The initial experiments performed on the Nova Petawatt Laser System were those associated with determining the nature of the electrons and x-rays in this relativistic regime especially those useful for advanced radiography sources suitable for diagnostic use in dense high-Z dynamic processes or as the driver of a relativistic electron source in the Fast-Ignitor Inertial Confinement fusion concept. The development of very large arrays of thermoluminescent detectors is detailed along with their response. The characteristic pattern of x-rays and their intensity is found from detailed analysis of the TLD detector array data. Peak intensities as high as 2 Rads at 1 meter were measured with these shielded TLD arrays. An average energy yield of x-rays of 11 Joules indicates a very large fraction of 45-55% of the laser energy is absorbed into relativistic electrons. The pattern of x-ray distribution lends insight to the initial relativistic electron distribution

  13. Transport characteristics of aerosol from urban point sources

    NASA Astrophysics Data System (ADS)

    Kunkel, Daniel; Lawrence, Mark G.; Kerkweg, Astrid; Tost, Holger; Jöckel, Patrick; Borrmann, Stephan

    2010-05-01

    Urban aerosols are an important source of regional and global air pollution. The local buildup, long-range transport, and dry and wet deposition of aerosols depend strongly on the aerosol size distribution and on the regional meteorological characteristics. We examine the characteristics of urban aerosol dispersion based on simulations of monodisperse passive aerosol tracers with sizes of 0.1, 1.0, 2.5, and 10.0 μm, performed with the global chemistry circulation model EMAC (ECHAM5-MESSy-Atmospheric-Chemistry). 39 point sources were selected for the analysis, originating from major population centers (MPCs) around the world. All tracers, one for each source and size, have the same total, constant emission flux, and undergo dry and wet aerosol deposition. Sensitivity simulations are performed in which either there is no activation of the aerosol as cloud condensation nuclei (CCN), or all aerosol is activated as CCN. Using the same constant emission rate for each MPC allows us to compare how different large point sources pollute the atmosphere and the surface on different horizontal scales. The transport and deposition of the aerosol tracers from each MPC are quantitatively compared by the application of metrics. The analysis focuses on: the efficiency of short- and long-range horizontal transport; the fraction of tracer transported to the upper troposphere; and the fractions which are dry or wet deposited. Smaller particles with longer lifetimes (two to 14 days) are more effective at polluting remote locations (horizontal and vertical) and are deposited mostly by scavenging, while larger particles, with shorter lifetimes (several hours to a couple of days) more effectively pollute the environment nearby their source, and are most strongly removed by dry deposition from the atmosphere. By means of considering the same emission for each city, the presentation provides a detailed view of how aerosol tracers disperse and deposit on different spatial scales, depending

  14. N-Methyl-D-Aspartate Receptor-Mediated Axonal Injury in Adult Rat Corpus Callosum

    PubMed Central

    Zhang, Jingdong; Liu, Jianuo; Fox, Howard S.; Xiong, Huangui

    2013-01-01

    Damage to white matter such as corpus callosum (CC) is a pathological characteristic in many brain disorders. Glutamate (Glut) excitotoxicity through AMPA receptors on oligodendrocyte (OL) was previously considered as a mechanism for white matter damage. Recent studies have shown that N-methyl-D-aspartate receptors (NMDARs) are expressed on myelin sheath of neonatal rat OL processes and that activation of these receptors mediated demyelization. Whether NMDARs are expressed in the adult CC and are involved in excitotoxic axonal injury remains to be determined. In this study, we demonstrate the presence of NMDARs in the adult rat CC and their distributions in myelinated nerve fibers and OL somata by means of immunocytochemical staining and Western blot. Incubation of the CC slices with Glut or NMDA induced axonal injury as revealed by analyzing amplitude of CC fiber compound action potentials (CAPs) and input–output response. Both Glut and NMDA decreased the CAP amplitude and input–output responses, suggesting an involvement of NMDARs in Glut- and NMDA-induced axonal injury. The involvement of NMDAR in Glut-induced axonal injury was further assayed by detection of β-amyloid precursor protein (β-APP) in the CC axonal fibers. Treatment of the CC slices with Glut resulted in β-APP accumulation in the CC fibers as detected by Western blot, reflecting an impairment of axonal transport function. This injurious effect of Glut on CC axonal transport was significantly blocked by MK801. Taken together, these results show that NMDARs are expressed in the adult CC and are involved in excitotoxic activity in adult CC slices in vitro. PMID:23161705

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

    PubMed

    Šmít, Daniel; Fouquet, Coralie; Pincet, Frédéric; Zapotocky, Martin; Trembleau, Alain

    2017-04-19

    While axon fasciculation plays a key role in the development of neural networks, very little is known about its dynamics and the underlying biophysical mechanisms. In a model system composed of neurons grown ex vivo from explants of embryonic mouse olfactory epithelia, we observed that axons dynamically interact with each other through their shafts, leading to zippering and unzippering behavior that regulates their fasciculation. Taking advantage of this new preparation suitable for studying such interactions, we carried out a detailed biophysical analysis of zippering, occurring either spontaneously or induced by micromanipulations and pharmacological treatments. We show that zippering arises from the competition of axon-axon adhesion and mechanical tension in the axons, and provide the first quantification of the force of axon-axon adhesion. Furthermore, we introduce a biophysical model of the zippering dynamics, and we quantitatively relate the individual zipper properties to global characteristics of the developing axon network. Our study uncovers a new role of mechanical tension in neural development: the regulation of axon fasciculation.

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

    PubMed Central

    Šmít, Daniel; Fouquet, Coralie; Pincet, Frédéric; Zapotocky, Martin; Trembleau, Alain

    2017-01-01

    While axon fasciculation plays a key role in the development of neural networks, very little is known about its dynamics and the underlying biophysical mechanisms. In a model system composed of neurons grown ex vivo from explants of embryonic mouse olfactory epithelia, we observed that axons dynamically interact with each other through their shafts, leading to zippering and unzippering behavior that regulates their fasciculation. Taking advantage of this new preparation suitable for studying such interactions, we carried out a detailed biophysical analysis of zippering, occurring either spontaneously or induced by micromanipulations and pharmacological treatments. We show that zippering arises from the competition of axon-axon adhesion and mechanical tension in the axons, and provide the first quantification of the force of axon-axon adhesion. Furthermore, we introduce a biophysical model of the zippering dynamics, and we quantitatively relate the individual zipper properties to global characteristics of the developing axon network. Our study uncovers a new role of mechanical tension in neural development: the regulation of axon fasciculation. DOI: http://dx.doi.org/10.7554/eLife.19907.001 PMID:28422009

  17. Extensive Acute Axonal Damage in Pediatric Multiple Sclerosis Lesions

    PubMed Central

    Pfeifenbring, Sabine; Bunyan, Reem F.; Metz, Imke; Röver, Christian; Huppke, Peter; Gärtner, Jutta; Lucchinetti, Claudia F.; Brück, Wolfgang

    2015-01-01

    Objective Axonal damage occurs early in multiple sclerosis (MS) and contributes to the degree of clinical disability. Children with MS more often show disabling and polyfocal neurological symptoms at disease onset than adults with MS. Thus, axonal damage may differ between pediatric and adult MS patients. Methods We analyzed axonal pathology in archival brain biopsy and autopsy samples from 19 children with early MS. Lesions were classified according to demyelinating activity and presence of remyelination. Axonal density and extent of acute axonal damage were assessed using Bielschowsky silver impregnation and immunohistochemistry for amyloid precursor protein (APP), respectively. Axonal injury was correlated with the inflammatory infiltrate as well as clinical characteristics. Results were compared with data from adult MS patients. Results Acute axonal damage was most extensive in early active demyelinating (EA) lesions of pediatric patients and correlated positively with the Expanded Disability Status Scale at attack leading to biopsy/autopsy. Comparison with 12 adult patients showed a 50% increase in the extent of acute axonal damage in EA lesions from children compared to adults, with the highest number of APP-positive spheroids found prior to puberty. The extent of acute axonal damage correlated positively with the number of lesional macrophages. Axonal density was reduced in pediatric lesions irrespective of the demyelinating activity or the presence of remyelination. Axonal reduction was similar between children and adults. Interpretation Our results provide evidence for more pronounced acute axonal damage in inflammatory demyelinating lesions from children compared to adults. PMID:25612167

  18. Contemplating Transport Characteristics by Augmenting the Length of Molecule

    NASA Astrophysics Data System (ADS)

    Kaur, Milanpreet; Sawhney, Ravinder Singh; Engles, Derick

    2013-11-01

    In this paper, we contemplated the transport characteristics of a single molecular device junction by augmenting the length of the molecule in the scattering region. The molecules considered here belongs to class of alkanedithiols (CnH2n+2S2). Specifically, we used a tight binding semi-empirical model to compute the transport characteristics of butanedithiol, pentanedithiol, hexanedithiol and heptanedithiol connected to semi-infinite gold electrodes through thiol anchoring elements. The exploration of transport properties of considered alkanes was completed for different bias voltages within the sphere of Keldysh's Non Equilibrium Green's Function (NEGF) and Extended Hückel Theory (EHT), for studying the self-consistent steady-state solution, analyzing the out-of-equilibrium electron distribution, and the behavior of the self-consistent potential. We perceived that the current and conductance retrenches with aggravation with the increase in length of the molecule with exhibition of single electron tunneling. We observed that the coupling regime shifts from strong coupling to weak for higher order alkanedithiols and the transmission is function of evenness or oddness of the carbon atoms forming an alkane.

  19. Characteristics of oesophageal bolus transport in patients with mild oesophagitis.

    PubMed

    Domingues, Gerson R; Winograd, Ron; Lemme, Eponina M; Lammert, Frank; Silny, Jiri; Matern, Siegfried; Nguyen, Huan N

    2005-03-01

    Patients with gastroesophageal reflux disease (GORD) frequently have oesophageal motility disturbance. However, detailed data about bolus transport characteristics in these patients are still lacking. In the present study the new technology of concurrent impedance manometry was applied for characterization of oesophageal motor function in patients with mild GORD. Oesophageal motility testing was performed in 25 patients with mild GORD (group 1) as compared to 25 healthy subjects (group 2) employing the technique of concurrent impedancometry and manometry. Oesophageal motility as well as patterns and parameters of bolus transport after the swallowing of saline or yogurt were analysed. According to manometry the velocity of the contraction wave was similar in both groups. Mid-distal contraction amplitude in group 1 was still in the normal range but significantly lower than in group 2 (57.4+/-4.5 mmHg vs 91.4+/-7.5 mmHg for saline, and 47+/-4.1 vs 80.7+/-9.4 mmHg for yogurt). According to impedance measurements, bolus transport was significantly slower (3.6+/-0.1 vs 4.0+/-0.1 cm/s for saline and 3.0+/-0.1 vs 3.2+/-0.1 cm/s for yogurt), and post-deglutitive impedance was significantly lower in group 1: 2110 omega+/-116 omega versus 2542 omega+/-152 omega (P<0.01) with saline and 1862 omega+/-108 omega versus 2348 omega+/-148 omega with yogurt (P<0.01). GORD patients showed several pathological bolus transport patterns, which were not observed in healthy subjects. Gastroesophageal liquid reflux was observed between the swallows. In patients with mild GORD concurrent impedancometry and manometry is sufficiently sensitive for the detection of minor oesophageal dysmotility. Several pathological features have been characterized including delayed bolus transport, impaired propulsive volume clearance, pathological transport patterns and pathological reflux patterns.

  20. Delayed Feedback Model of Axonal Length Sensing

    PubMed Central

    Karamched, Bhargav R.; Bressloff, Paul C.

    2015-01-01

    A fundamental question in cell biology is how the sizes of cells and organelles are regulated at various stages of development. Size homeostasis is particularly challenging for neurons, whose axons can extend from hundreds of microns to meters (in humans). Recently, a molecular-motor-based mechanism for axonal length sensing has been proposed, in which axonal length is encoded by the frequency of an oscillating retrograde signal. In this article, we develop a mathematical model of this length-sensing mechanism in which advection-diffusion equations for bidirectional motor transport are coupled to a chemical signaling network. We show that chemical oscillations emerge due to delayed negative feedback via a Hopf bifurcation, resulting in a frequency that is a monotonically decreasing function of axonal length. Knockdown of either kinesin or dynein causes an increase in the oscillation frequency, suggesting that the length-sensing mechanism would produce longer axons, which is consistent with experimental findings. One major prediction of the model is that fluctuations in the transport of molecular motors lead to a reduction in the reliability of the frequency-encoding mechanism for long axons. PMID:25954897

  1. Emerging brain morphologies from axonal elongation

    PubMed Central

    Holland, Maria A.; Miller, Kyle E.; Kuhl, Ellen

    2015-01-01

    Understanding the characteristic morphology of our brain remains a challenging, yet important task in human evolution, developmental biology, and neurosciences. Mathematical modeling shapes our understanding of cortical folding and provides functional relations between cortical wavelength, thickness, and stiffness. Yet, current mathematical models are phenomenologically isotropic and typically predict non-physiological, periodic folding patterns. Here we establish a mechanistic model for cortical folding, in which macroscopic changes in white matter volume are a natural consequence of microscopic axonal growth. To calibrate our model, we consult axon elongation experiments in chick sensory neurons. We demonstrate that a single parameter, the axonal growth rate, explains a wide variety of in vitro conditions including immediate axonal thinning and gradual thickness restoration. We embed our axonal growth model into a continuum model for brain development using axonal orientation distributions motivated by diffusion spectrum imaging. Our simulations suggest that white matter anisotropy - as an emergent property from directional axonal growth - intrinsically induces symmetry breaking, and predicts more physiological, less regular morphologies with regionally varying gyral wavelengths and sulcal depths. Mechanistic modeling of brain development could establish valuable relationships between brain connectivity, brain anatomy, and brain function. PMID:25824370

  2. Early posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport.

    PubMed

    Nixon, R A; Lewis, S E; Dahl, D; Marotta, C A; Drager, U C

    1989-03-01

    We have characterized stages in the posttranslational processing of the three neurofilament subunits, High (NF-H), Middle (NF-M), and Low (NF-L), in retinal ganglion cells in vivo during the interval between synthesis in cell bodies within the retina and appearance of these polypeptides in axons at the level of the optic nerve (optic axons). Neurofilament proteins pulse-labeled by injecting mice intravitreally with [35S]methionine or [32P]orthophosphate, were isolated from Triton-soluble and Triton-insoluble fractions of the retina or optic axons by immunoprecipitation or immunoaffinity chromatography. Within 2 h after [35S]methionine injection, the retina contained neurofilament-immunoreactive radiolabeled proteins with apparent molecular weights of 160, 139, and 70 kDa, which co-migrated with subunits of axonal neurofilaments that were dephosphorylated in vitro with alkaline phosphatase. The two larger polypeptides were not labeled with [32P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M. About 75% of the subunits were Triton-insoluble by 2 h after isotope injection, and this percentage increased to 98% by 6 h. Labeled neurofilament polypeptides appeared in optic axons as early as 2 h after injection. These subunits exhibited apparent molecular weights of 160, 139, and 70 kDa and were Triton-insoluble. The time of appearance of fully modified polypeptide forms differed for each subunit (2 h for NF-L, 6-18 h for NF-M, 18-24 h for NF-H) and was preceded by the transient appearance of intermediate forms. The modified radiolabeled subunits in optic axons 3 days after synthesis were heavily labeled with [32P]orthophosphate and exhibited the same apparent molecular weights as subunits of axonal neurofilaments (70 kDa, 145 and 140 kDa, and 195-210 kDa, respectively). Whole mounts of retina immunostained with monoclonal antibodies against NF-H in different states of phosphorylation demonstrated a transition from non

  3. Characteristics of soil radon transport in different geological formations

    NASA Astrophysics Data System (ADS)

    Ershaidat, N. M.; Al-Bataina, B. A.; Al-Shereideh, S. A.

    2008-07-01

    Soil radon concentration levels in Deir Abu-Said District, Irbid, Jordan were measured for several depths using CR-39 detectors, in the summer 2004 for six geological formations, namely, Wadi umm ghudran (WG), Wadi esSir “massive” limestone, Amman silicified limestone (ASL), Al-Hisa phosphatic limestone (AHP), Muwaqqar chalky-marl (MCM), and Basalt. Using a model (Yakovleva in Ann Geophys 48(1):195 198, 2005) based on the solution of the diffusion equation in the quasi-homogenous approximation, the characteristics of radon transport were calculated. Radon flux density from the Earth’s surface, the depth Z eq, at which the equilibrium value of soil radon concentration is reached and the convective radon flux velocity ( v) for the different soils are calculated and found to be consistent with similar values presented elsewhere. Calculations indicate that the soil covering WG has a low radon risk while, on the contrary, AHP has a higher radon risk as expected, since AHP has higher content of uranium. The other formations have intermediate values. The results of the present study confirm the statement by Yakovleva (Ann Geophys 48(1):195 198, 2005) that two measurements suffice in order to estimate the characteristics of soil radon transport.

  4. Unique Function of Kinesin Kif5A in Localization of Mitochondria in Axons

    PubMed Central

    Campbell, Philip D.; Shen, Kimberle; Sapio, Matthew R.; Glenn, Thomas D.; Talbot, William S.

    2014-01-01

    Mutations in Kinesin proteins (Kifs) are linked to various neurological diseases, but the specific and redundant functions of the vertebrate Kifs are incompletely understood. For example, Kif5A, but not other Kinesin-1 heavy-chain family members, is implicated in Charcot-Marie-Tooth disease (CMT) and Hereditary Spastic Paraplegia (HSP), but the mechanism of its involvement in the progressive axonal degeneration characteristic of these diseases is not well understood. We report that zebrafish kif5Aa mutants exhibit hyperexcitability, peripheral polyneuropathy, and axonal degeneration reminiscent of CMT and HSP. Strikingly, although kif5 genes are thought to act largely redundantly in other contexts, and zebrafish peripheral neurons express five kif5 genes, kif5Aa mutant peripheral sensory axons lack mitochondria and degenerate. We show that this Kif5Aa-specific function is cell autonomous and is mediated by its C-terminal tail, as only Kif5Aa and chimeric motors containing the Kif5Aa C-tail can rescue deficits. Finally, concurrent loss of the kinesin-3, kif1b, or its adaptor kbp, exacerbates axonal degeneration via a nonmitochondrial cargo common to Kif5Aa. Our results shed light on Kinesin complexity and reveal determinants of specific Kif5A functions in mitochondrial transport, adaptor binding, and axonal maintenance. PMID:25355224

  5. Axonal GABAA receptors.

    PubMed

    Trigo, Federico F; Marty, Alain; Stell, Brandon M

    2008-09-01

    Type A GABA receptors (GABA(A)Rs) are well established as the main inhibitory receptors in the mature mammalian forebrain. In recent years, evidence has accumulated showing that GABA(A)Rs are prevalent not only in the somatodendritic compartment of CNS neurons, but also in their axonal compartment. Evidence for axonal GABA(A)Rs includes new immunohistochemical and immunogold data: direct recording from single axonal terminals; and effects of local applications of GABA(A)R modulators on action potential generation, on axonal calcium signalling, and on neurotransmitter release. Strikingly, whereas presynaptic GABA(A)Rs have long been considered inhibitory, the new studies in the mammalian brain mostly indicate an excitatory action. Depending on the neuron that is under study, axonal GABA(A)Rs can be activated by ambient GABA, by GABA spillover, or by an autocrine action, to increase either action potential firing and/or transmitter release. In certain neurons, the excitatory effects of axonal GABA(A)Rs persist into adulthood. Altogether, axonal GABA(A)Rs appear as potent neuronal modulators of the mammalian CNS.

  6. Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

    PubMed Central

    Yoon, Byung C.; Jung, Hosung; Dwivedy, Asha; O'Hare, Catherine M.; Zivraj, Krishna H.; Holt, Christine E.

    2012-01-01

    Summary Local protein synthesis plays a key role in regulating stimulus-induced responses in dendrites and axons. Recent genome-wide studies have revealed that thousands of different transcripts reside in these distal neuronal compartments, but identifying those with functionally significant roles presents a challenge. We performed an unbiased screen to look for stimulus-induced, protein synthesis-dependent changes in the proteome ofXenopus retinal ganglion cell (RGC) axons. The intermediate filament protein lamin B2 (LB2), normally associated with the nuclear membrane, was identified as an unexpected major target. Axonal ribosome immunoprecipitation confirmed translation of lb2 mRNA in vivo. Inhibition of lb2 mRNA translation in axons in vivo does not affect guidance but causes axonal degeneration. Axonal LB2 associates with mitochondria, and LB2-deficient axons exhibit mitochondrial dysfunction and defects in axonal transport. Our results thus suggest that axonally synthesized lamin B plays a crucial role in axon maintenance by promoting mitochondrial function. PMID:22341447

  7. Atomic Force Microscopy Reveals Important Differences in Axonal Resistance to Injury

    PubMed Central

    Magdesian, Margaret H.; Sanchez, Fernando S.; Lopez, Monserratt; Thostrup, Peter; Durisic, Nela; Belkaid, Wiam; Liazoghli, Dalinda; Grütter, Peter; Colman, David R.

    2012-01-01

    Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 ± 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 ± 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons. PMID:22947856

  8. Can injured adult CNS axons regenerate by recapitulating development?

    PubMed

    Hilton, Brett J; Bradke, Frank

    2017-10-01

    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.

  9. Characteristics and Possible Functions of Mitochondrial Ca2+ Transport Mechanisms

    PubMed Central

    Gunter, Thomas E.; Sheu, Shey-Shing

    2009-01-01

    Mitochondria produce around 92% of the ATP used in the typical animal cell by oxidative phosphorylation using energy from their electrochemical proton gradient. Intramitochondrial free Ca2+ concentration ([Ca2+]m) has been found to be an important component of control of the rate of this ATP production. In addition, [Ca2+]m also controls the opening of a large pore in the inner mitochondrial membrane, the permeability transition pore (PTP), which plays a role in mitochondrial control of programmed cell death or apoptosis. Therefore, [Ca2+]m can control whether the cell has sufficient ATP to fulfill its functions and survive or is condemned to death. Ca2+ is also one of the most important second messengers within the cytosol, signaling changes in cellular response through Ca2+ pulses or transients. Mitochondria can also sequester Ca2+ from these transients so as to modify the shape of Ca2+ signaling transients or control their location within the cell. All of this is controlled by the action of four or five mitochondrial Ca2+ transport mechanisms and the PTP. The characteristics of these mechanisms of Ca2+ transport and a discussion of how they might function are described in this paper. PMID:19161975

  10. Kinesin-1 transport reductions enhance human tau hyperphosphorylation, aggregation and neurodegeneration in animal models of tauopathies.

    PubMed

    Falzone, Tomás L; Gunawardena, Shermali; McCleary, David; Reis, Gerald F; Goldstein, Lawrence S B

    2010-11-15

    Neurodegeneration induced by abnormal hyperphosphorylation and aggregation of the microtubule-associated protein tau defines neurodegenerative tauopathies. Destabilization of microtubules by loss of tau function and filament formation by toxic gain of function are two mechanisms suggested for how abnormal tau triggers neuronal loss. Recent experiments in kinesin-1 deficient mice suggested that axonal transport defects can initiate biochemical changes that induce activation of axonal stress kinase pathways leading to abnormal tau hyperphosphorylation. Here we show using Drosophila and mouse models of tauopathies that reductions in axonal transport can exacerbate human tau protein hyperphosphorylation, formation of insoluble aggregates and tau-dependent neurodegeneration. Together with previous work, our results suggest that non-lethal reductions in axonal transport, and perhaps other types of minor axonal stress, are sufficient to induce and/or accelerate abnormal tau behavior characteristic of Alzheimer's disease and other neurodegenerative tauopathies.

  11. Astrocytes Block Axonal Regeneration in Mammals by Activating the Physiological Stop Pathway

    NASA Astrophysics Data System (ADS)

    Liuzzi, Francis J.; Lasek, Raymond J.

    1987-08-01

    Regenerating sensory axons in the dorsal roots of adult mammals are stopped at the junction between the root and spinal cord by reactive astrocytes. Do these cells stop axonal elongation by activating the physiological mechanisms that normally operate to stop axons during development, or do they physically obstruct the elongating axons? In order to distinguish these possibilities, the cytology of the axon tips of regenerating axons that were stopped by astrocytes was compared with the axon tips that were physically obstructed at a cul-de-sac produced by ligating a peripheral nerve. The terminals of the physically obstructed axon tips were distended with neurofilaments and other axonally transported structures that had accumulated when the axons stopped elongating. By contrast, neurofilaments did not accumulate in the tips of regenerating axons that were stopped by spinal cord astrocytes at the dorsal root transitional zone. These axo-glial terminals resembled the terminals that axons make on target neurons during normal development. On the basis of these observations, astrocytes appear to stop axons from regenerating in the mammalian spinal cord by activating the physiological stop pathway that is built into the axon and that normally operates when axons form stable terminals on target cells.

  12. Characteristics of atmospheric transport into the Antarctic troposphere

    NASA Astrophysics Data System (ADS)

    Stohl, A.; Sodemann, H.

    2010-01-01

    We have developed a 5.5 year climatology of atmospheric transport into the Antarctic troposphere, which uses the same data set and methods as described in a recent study for the Arctic. This allows direct comparisons of transport properties for the two polar regions. The climatology is based on a simulation with the Lagrangian particle dispersion model FLEXPART, where the model atmosphere was globally filled with particles. Transport characteristics as well as emission sensitivities were derived from 6 hourly particle positions. We found that the probability for near-surface air to originate from the stratosphere on a time scale of 10 days is an order of magnitude higher near the South Pole than near the North Pole, a result of higher topography and descent that partly compensates for the flow of air down the Antarctic Plateau with the katabatic winds. The stratospheric influence is largest in fall, which is opposite to the seasonality in the Arctic. Stratospheric influence is much smaller over the shelf ice regions and in a band around Antarctica. The average time for which air near the surface has been exposed to continuous darkness in July (continuous light in January) is longest over the Ronne Ice Shelf and Ross Ice Shelf at ˜11 days (20 days). We calculated how sensitive Antarctic air masses are to emission input up to 30 days before arriving in Antarctica if removal processes are ignored. The emission sensitivity shows strong meridional gradients and, as a result, is generally low over South America, Africa, and Australia. For a 10 day time scale, the largest emission sensitivities over these continents are 1-2 orders of magnitude smaller than over Eurasia for transport to the Arctic, showing that foreign continents have a much smaller potential to pollute the Antarctic than the Arctic troposphere. Emission sensitivities and derived black carbon (BC) source contributions over South America, Africa, and Australia are substantially (a factor 10 for Africa

  13. Axons take a dive

    PubMed Central

    Tong, Cheuk Ka; Cebrián-Silla, Arantxa; Paredes, Mercedes F; Huang, Eric J; García-Verdugo, Jose Manuel; Alvarez-Buylla, Arturo

    2015-01-01

    In the walls of the lateral ventricles of the adult mammalian brain, neural stem cells (NSCs) and ependymal (E1) cells share the apical surface of the ventricular–subventricular zone (V–SVZ). In a recent article, we show that supraependymal serotonergic (5HT) axons originating from the raphe nuclei in mice form an extensive plexus on the walls of the lateral ventricles where they contact E1 cells and NSCs. Here we further characterize the contacts between 5HT supraependymal axons and E1 cells in mice, and show that suprependymal axons tightly associated to E1 cells are also present in the walls of the human lateral ventricles. These observations raise interesting questions about the function of supraependymal axons in the regulation of E1 cells. PMID:26413556

  14. Thiamine transport across the rat intestine. I. Normal characteristics.

    PubMed

    Hoyumpa, A M; Middleton, H M; Wilson, F A; Schenker, S

    1975-05-01

    The characteristics of normal thiamine transport across the intestine were studied in rats using intact intestinal loops and everted jejunal segments. In vivo studies with [35-S]-thiamine hydrochloride revealed, in all segments of small intestine, saturation kinetics for low thiamine concentrations (0.06 to 1.5 muM), but a linear relationship between high concentrations (2 to 560 muM) and absorption. Moreover, in vitro studies of net transmural flux using everted jejunal sacs demonstrated movement of [14-C]-thiamine hydrochloride against a concentration gradient only when low, but not when high, thiamine concentration was used, so that the serosal to mucosal ratio became significantly greater than the initial value of one. Pyrithiamine, 2 muM, dinitrophenol, 200 muM, norethylmaleimide, 100 muM, and ouabain, 10 muM, reduced the net transmural flux of 0.2 muM thiamine. In contrast, these inhibitors had no effect on 20 muM thiamine. When unidirectional flux across the jejunum was measured, saturation kinetics was again demonstrated for low thiamine concentrations. This phenomenon, however, was abolished by the addition of pyrithiamine, which exerted competitive inhibition on thiamine absorption. Anoxia and sodium lack reduced intestinal uptake of 0.5 muM thiamine to 58% and 74% of normal, respectively, but did not affect uptake of 50 muM thiamine. Lowering the marked with low thiamine concentrations (O10, 1.648) than with high concentration (Q10, 1.127). Stirring of the water layer reduced Km to 59% of unstirred value, while Vmax and permeability coefficient remained unchanged. Finally, movement of low concentration thiamine against an electrical gradient was observed under conditions of electrical short circuiting and zero potential difference. In contrast, no such effect was seen with high concentrations. These studies suggest that there exists in the rat a dual system of intestinal thiamine transport. At low concentrations, thiamine is absorbed by an active process

  15. The Number of Alphaherpesvirus Particles Infecting Axons and the Axonal Protein Repertoire Determines the Outcome of Neuronal Infection

    PubMed Central

    Koyuncu, Orkide O.; Song, Ren; Greco, Todd M.; Cristea, Ileana M.

    2015-01-01

    ABSTRACT Infection by alphaherpesviruses invariably results in invasion of the peripheral nervous system (PNS) and establishment of either a latent or productive infection. Infection begins with long-distance retrograde transport of viral capsids and tegument proteins in axons toward the neuronal nuclei. Initial steps of axonal entry, retrograde transport, and replication in neuronal nuclei are poorly understood. To better understand how the mode of infection in the PNS is determined, we utilized a compartmented neuron culturing system where distal axons of PNS neurons are physically separated from cell bodies. We infected isolated axons with fluorescent-protein-tagged pseudorabies virus (PRV) particles and monitored viral entry and transport in axons and replication in cell bodies during low and high multiplicities of infection (MOIs of 0.01 to 100). We found a threshold for efficient retrograde transport in axons between MOIs of 1 and 10 and a threshold for productive infection in the neuronal cell bodies between MOIs of 1 and 0.1. Below an MOI of 0.1, the viral genomes that moved to neuronal nuclei were silenced. These genomes can be reactivated after superinfection by a nonreplicating virus, but not by a replicating virus. We further showed that viral particles at high-MOI infections compete for axonal proteins and that this competition determines the number of viral particles reaching the nuclei. Using mass spectrometry, we identified axonal proteins that are differentially regulated by PRV infection. Our results demonstrate the impact of the multiplicity of infection and the axonal milieu on the establishment of neuronal infection initiated from axons. PMID:25805728

  16. Concepts for regulation of axon integrity by enwrapping glia

    PubMed Central

    Beirowski, Bogdan

    2013-01-01

    Long axons and their enwrapping glia (EG; Schwann cells (SCs) and oligodendrocytes (OLGs)) 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 SCs and OLGs . 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 EG’s non-cell autonomous impact on axon-integrity. A particular focus is placed on emerging evidence suggesting that EG 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 nervous system and central nervous system (PNS and CNS). PMID:24391540

  17. Sediment-transport characteristics of Cane Creek, Lauderdale County, Tennessee

    USGS Publications Warehouse

    Carey, W.P.

    1993-01-01

    An investigation of the sediment-transport characteristics of Cane Creek in Lauderdale County, Tennessee, was conducted from 1985-88 to evaluate the potential for channel erosion induced by modifications (realignment and enlargement) and the potential ability of different flows to move bed and bank stabilizing material. Frequently occurring flows in Cane Creek are capable of moving sand-size material (0.0625 - 4.0 millimeters). During floods that equal or exceed the 2-year flood, Cane Creek is capable of moving very coarse gravel (32 - 64 millimeters). Boundary-shear values at bridges, where flow contractions occur, correspond to critical diameters in excess of 100 millimeters. Thus, the areas near bridges, where channel stability is most critical, are the areas where erosive power is greatest. Deepening and widening of Cane Creek has exposed large areas of channel boundary that are a significant source of raindrop-detached sediment during the early stages of a storm before stream flow increases signifi- cantly. This causes suspended-sediment concentration to peak while the flow hydrograph is just beginning to rise. For basins like Cane Creek, where runoff events commonly last less than a day and where variation in discharge and sediment concentrations are large, an estimate of sediment yield based on periodic observations of instantaneous values is subject to considerable uncertainty.

  18. Building and maintaining the axon initial segment

    PubMed Central

    Grubb, Matthew S.; Burrone, Juan

    2011-01-01

    The axon initial segment is a unique neuronal subregion involved in the initiation of action potentials and in the control of axonal identity. Recent work has helped our understanding of how this specialised structure develops, not least in identifying possible mechanisms leading to the localisation of the AIS’s master organiser protein, ankyrin-G. The most exciting current work, however, focuses on later aspects of AIS function and plasticity. Recent studies have shown that the AIS is subdivided into distinct structural and functional domains, have demonstrated how the AIS acts as a cytoplasmic barrier for axonal transport, and have discovered that the AIS can be surprisingly plastic in its responses to alterations in neuronal activity. PMID:20537529

  19. Demyelination and axonal preservation in a transgenic mouse model of Pelizaeus-Merzbacher disease

    PubMed Central

    Edgar, Julia M; McCulloch, Mailis C; Montague, Paul; Brown, Angus M; Thilemann, Sebastian; Pratola, Laura; Gruenenfelder, Fredrik I; Griffiths, Ian R; Nave, Klaus-Armin

    2010-01-01

    It is widely thought that demyelination contributes to the degeneration of axons and, in combination with acute inflammatory injury, is responsible for progressive axonal loss and persistent clinical disability in inflammatory demyelinating disease. In this study we sought to characterize the relationship between demyelination, inflammation and axonal transport changes using a Plp1-transgenic mouse model of Pelizaeus-Merzbacher disease. In the optic pathway of this non-immune mediated model of demyelination, myelin loss progresses from the optic nerve head towards the brain, over a period of months. Axonal transport is functionally perturbed at sites associated with local inflammation and ‘damaged’ myelin. Surprisingly, where demyelination is complete, naked axons appear well preserved despite a significant reduction of axonal transport. Our results suggest that neuroinflammation and/or oligodendrocyte dysfunction are more deleterious for axonal health than demyelination per se, at least in the short term. PMID:20091761

  20. Axonal maintenance, glia, exosomes, and heat shock proteins

    PubMed Central

    Tytell, Michael; Lasek, Raymond J.; Gainer, Harold

    2016-01-01

    Of all cellular specializations, the axon is especially distinctive because it is a narrow cylinder of specialized cytoplasm called axoplasm with a length that may be orders of magnitude greater than the diameter of the cell body from which it originates. Thus, the volume of axoplasm can be much greater than the cytoplasm in the cell body. This fact raises a logistical problem with regard to axonal maintenance. Many of the components of axoplasm, such as soluble proteins and cytoskeleton, are slowly transported, taking weeks to months to travel the length of axons longer than a few millimeters after being synthesized in the cell body. Furthermore, this slow rate of supply suggests that the axon itself might not have the capacity to respond fast enough to compensate for damage to transported macromolecules. Such damage is likely in view of the mechanical fragility of an axon, especially those innervating the limbs, as rapid limb motion with high impact, like running, subjects the axons in the limbs to considerable mechanical force. Some researchers have suggested that local, intra-axonal protein synthesis is the answer to this problem. However, the translational state of axonal RNAs remains controversial. We suggest that glial cells, which envelop all axons, whether myelinated or not, are the local sources of replacement and repair macromolecules for long axons. The plausibility of this hypothesis is reinforced by reviewing several decades of work on glia-axon macromolecular transfer, together with recent investigations of exosomes and other extracellular vesicles, as vehicles for the transmission of membrane and cytoplasmic components from one cell to another. PMID:26962444

  1. Axonal isoforms of myosin-I.

    PubMed

    Lund, Linda M; Machado, Victor M; McQuarrie, Irvine G

    2005-05-13

    We have examined spinal motor neurons in Sprague-Dawley rats to further characterize a mechanoenzyme, myosin-Igamma (myr4), which is found in high concentration during axon tract formation in neonates. We raised an antibody to myr4 and made riboprobes for in situ hybridization. Myr4 mRNA was abundant in spinal cord motor neurons (particularly during axon regrowth). Nerves undergoing Wallerian degeneration (from a crush 7 days earlier) showed anti-myr4 labeling of the axolemma and SER--after microtubules, neurofilaments, and F-actin had already been degraded--which is consistent with a described lipid-binding domain in the tail region of myosin-Is. Newly synthesized myr4 was carried in axons by the slow component (SC) of axonal transport at 1-8 mm/day, whereas, none was carried by the fast component (FC). We conclude that SC delivers myr4 to the cytoplasmic surfaces of stationary axonal membranes (SER and axolemma). This positioning would anchor the tail domain of myr4 and leave the catalytic head domain free to interact with F-actin.

  2. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  3. Mechanical Properties of Axons

    NASA Astrophysics Data System (ADS)

    Bernal, Roberto; Pullarkat, Pramod A.; Melo, Francisco

    2007-07-01

    The mechanical response of PC12 neurites under tension is investigated using a microneedle technique. Elastic response, viscoelastic relaxation, and active contraction are observed. The mechanical model proposed by Dennerll et al. [J. Cell Biol. 109, 3073 (1989).JCLBA30021-952510.1083/jcb.109.6.3073], which involves three mechanical devices—a stiff spring κ coupled with a Voigt element that includes a less stiff spring k and a dashpot γ—has been improved by adding a new element to describe the main features of the contraction of axons. This element, which represents the action of molecular motors, acts in parallel with viscous forces defining a global tension response of axons T against elongation rates δ˙k. Under certain conditions, axons show a transition from a viscoelastic elongation to active contraction, suggesting the presence of a negative elongation rate sensitivity in the curve T vs δ˙k.

  4. Exclusion of Integrins from CNS Axons Is Regulated by Arf6 Activation and the AIS

    PubMed Central

    Franssen, Elske H. P.; Zhao, Rong-Rong; Koseki, Hiroaki; Kanamarlapudi, Venkateswarlu; Hoogenraad, Casper C.

    2015-01-01

    Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking. PMID:26019348

  5. Transport characteristics of isorhamnetin across intestinal Caco-2 cell monolayers and the effects of transporters on it.

    PubMed

    Duan, Jingze; Xie, Yan; Luo, Huilin; Li, Guowen; Wu, Tao; Zhang, Tong

    2014-04-01

    Flavonoid isorhamnetin occurs in various plants and herbs, and demonstrates various biological effects in humans. This work will clarify the isorhamnetin absorption mechanism using the Caco-2 monolayer cell model. The isorhamnetin transport characteristics at different concentrations, pHs, temperatures, tight junctions and potential transporters were systemically investigated. Isorhamnetin was poorly absorbed by both passive diffusion and active transport mechanisms. Both trans- and paracellular pathways were involved during isorhamnetin transport. Active transport under an ATP-dependent transport mechanism was mediated by the organic anion transporting peptide (OATP); isorhamnetin's permeability from the apical to the basolateral side significantly decreased after estrone-3-sulfate was added (p<0.01). Efflux transporters, P-glycoproteins (P-gp), breast cancer resistance proteins (BCRP) and multidrug resistance proteins (MRPs) participated in the isorhamnetin transport process. Among them, the MRPs (especially MRP2) were the main efflux transporters for isorhamnetin; transport from the apical to the basolateral side increased 10.8-fold after adding an MRP inhibitor (MK571). This study details isorhamnetin's cellular transport and elaborates isorhamnetin's absorption mechanisms to provide a foundation for further studies.

  6. KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons.

    PubMed

    Lo, K Y; Kuzmin, A; Unger, S M; Petersen, J D; Silverman, M A

    2011-03-24

    Dense-core vesicles (DCVs) are responsible for transporting, processing, and secreting neuropeptide cargos that mediate a wide range of biological processes, including neuronal development, survival, and learning and memory. DCVs are synthesized in the cell body and are transported by kinesin motor proteins along microtubules to pre- and postsynaptic release sites. Due to the dependence on kinesin-based transport, we sought to determine if the kinesin-3 family member, KIF1A, transports DCVs in primary cultured hippocampal neurons, as has been described for invertebrate neurons. Two-color, live-cell imaging showed that the DCV markers, chromogranin A-RFP and BDNF-RFP, move together with KIF1A-GFP in both the anterograde and retrograde directions. To demonstrate a functional role for KIF1A in DCV transport, motor protein expression in neurons was reduced using RNA interference (shRNA). Fluorescently tagged DCV markers showed a significant reduction in organelle flux in cells expressing shRNA against KIF1A. The transport of cargo driven by motors other than KIF1A, including mitochondria and the transferrin receptor, was unaffected in KIF1A shRNA expressing cells. Taken together, these data support a primary role for KIF1A in the anterograde transport of DCVs in mammalian neurons, and also provide evidence that KIF1A remains associated with DCVs during retrograde DCV transport. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

  7. Peritoneal transport characteristics with glucose polymer based dialysate.

    PubMed

    Ho-dac-Pannekeet, M M; Schouten, N; Langendijk, M J; Hiralall, J K; de Waart, D R; Struijk, D G; Krediet, R T

    1996-09-01

    it remained unchanged during the dwell. In contrast, D/PNa+ of 1.36% glucose increased during the dwell, whereas D/PNa+ decreased with 3.86% glucose until 60 minutes, followed by a subsequent increase. The ultrafiltration coefficient (UFC) of the total peritoneal membrane was assessed using 3.86% glucose (0.18 +/- 0.04 ml/min/mm Hg), and the UFC of the small pores was assessed using icodextrin (0.06 +/- 0.008 ml/min/mm Hg). The difference between these represented the UFC through the transcellular pores, which averaged 50.5% of the total UFC, but with a very wide range (0 to 85%). An inverse relation existed between the duration of CAPD treatment and the total ultrafiltration coefficient (r = -0.68, P < 0.04), which could be attributed to a lower UFC of the transcellular pores in long-term patients (r = -0.66, P < 0.05), but not to the UFC of the small pores (r = -0.48, NS). The TCUFRo-60 min through the transcellular pores correlated with the sodium gradient, corrected for diffusion, in the first hour of the dwell (r = 0.69, P < 0.04), indicating that both parameters indeed measure transcellular water transport. It can be concluded that the glucose polymer solution induced sustained ultrafiltration and had no effect on peritoneal membrane characteristics. In addition, the results of the present study support the hypothesis that the glucose polymer solutions exerts its osmotic pressure across intercellular pores with radii of about 40 A. This leads to increased clearances of low molecular weight proteins such as beta 2m that are transported through these pores without sieving of Na+. The latter, as found during 3.86% glucose dialysate, is probably caused by transcellular water transport. The transcellular water transport accounted for 50% of the total ultrafiltration with glucose based dialysis solutions. It was lower in long-term CAPD patients.

  8. Neurofilament gene expression: a major determinant of axonal caliber

    SciTech Connect

    Hoffman, P.N.; Cleveland, D.W.; Griffin, J.W.; Landes, P.W.; Cowan, N.J.; Price, D.L.

    1987-05-01

    Within the wide spectrum of axonal diameters occurring in mammalian nerve fibers, each class of neurons has a relatively restricted range of axonal calibers. The control of caliber has functional significance because diameter is the principal determinant of conduction velocity in myelinated nerve fibers. Previous observations support the hypothesis that neurofilaments (NF) are major intrinsic determinants of axonal caliber in large myelinated nerve fibers. Following interruption of axons (axotomy) by crushing or cutting a peripheral nerve, caliber is reduced in the proximal axonal stumps, which extend from the cell bodies to the site of axotomy. This reduction in axonal caliber in the proximal stumps is associated with a selective diminution in the amount of NF protein undergoing slow axonal transport in these axons, with a decrease in axonal NF content, and with reduced conduction velocity. The present report demonstrates that changes in axonal caliber after axotomy correlate with a selective alteration in NF gene expression. Hybridization with specific cDNAs was used to measure levels of mRNA encoding the 68-kDa neurofilament protein (NF68), ..beta..-tubulin, and actin in lumbar sensory neurons of rat at various times after crushing the sciatic nerve. Between 4 and 42 days after axotomy by nerve crush, the levels of NF68 mRNA were reduced 2- to 3-fold. At the same times, the levels of tubulin and actin mRNAs were increased several-fold. These findings support the hypothesis that the expression of a single set of neuron-specific genes (encoding NF) directly determines axonal caliber, a feature neuronal morphology with important consequences for physiology and behavior.

  9. Initiating and Growing an Axon

    PubMed Central

    Polleux, F.; Snider, William

    2010-01-01

    The ability of neurons to form a single axon and multiple dendrites underlies the directional flow of information transfer in the central nervous system. Dendrites and axons are molecularly and functionally distinct domains. Dendrites integrate synaptic inputs, triggering the generation of action potentials at the level of the soma. Action potentials then propagate along the axon, which makes presynaptic contacts onto target cells. This article reviews what is known about the cellular and molecular mechanisms underlying the ability of neurons to initiate and extend a single axon during development. Remarkably, neurons can polarize to form a single axon, multiple dendrites, and later establish functional synaptic contacts in reductionist in vitro conditions. This approach became, and remains, the dominant model to study axon initiation and growth and has yielded the identification of many molecules that regulate axon formation in vitro ( Dotti et al. 1988). At present, only a few of the genes identified using in vitro approaches have been shown to be required for axon initiation and outgrowth in vivo. In vitro, axon initiation and elongation are largely intrinsic properties of neurons that are established in the absence of relevant extracellular cues. However, the importance of extracellular cues to axon initiation and outgrowth in vivo is emerging as a major theme in neural development ( Barnes and Polleux 2009). In this article, we focus our attention on the extracellular cues and signaling pathways required in vivo for axon initiation and axon extension. PMID:20452947

  10. Characteristics of microseisms recorded by the Earthscope Transportable Array

    NASA Astrophysics Data System (ADS)

    Sufri, Oner

    I analyzed the characteristics of microseisms recorded in the United States by Earthscope Transportable Array (TA) broadband stations during the calendar year of 2009 and a 19-day period of October-November 2012. I used eigen-decomposition of spectral covariance matrices to extract power and polarization information for each hour of data recorded at each seismometer. For the continuous data from 2009, I generated array-averaged spectrograms and geographical animations to locate individual microseisms. Then, I grouped and cataloged those microseisms according to their initiation time, duration, peak power, average power, dominant period, variation in their period content, degree of polarization, and their azimuths obtained from polarization ellipsoids. Over 78 distinct microseismic events were identified and grouped into four different types. The longest duration microseismic signal occurred in the month of December, 2009, for more than 280 hours and was associated with the propagation of two storms: one from the Gulf of Alaska region and another from the Newfoundland region. The most powerful signal was also recorded in the same month with an average peak period near 6-sec on December 28-31, 2009, and resulted from wave action associated with two different unnamed storms in the East-Central Pacific and North Atlantic oceans. The seismic observations were compared to excitation predictions computed with the power spectral density of the equivalent pressure generated by ocean gravity waves using the WAVEWATCH-III ocean wave model from the French Research Institute for Exploitation of the Sea (Ifremer). Microseismic excitation predictions were calculated both with and without coastline reflections. I also processed continuous TA seismic data from 17 October-4 November, 2012, coinciding with the passage of Hurricane Sandy. I determined and tracked locations of microseisms as the hurricane propagated from South to North along the U.S. Atlantic coast. I found that the

  11. Axons provide the secretory machinery for trafficking of voltage-gated sodium channels in peripheral nerve

    PubMed Central

    González, Carolina; Cánovas, José; Fresno, Javiera; Couve, Eduardo; Court, Felipe A.; Couve, Andrés

    2016-01-01

    The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and are mostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodium channels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retention-release methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons. PMID:26839409

  12. Characteristics and regulation of proline transport in cultured glioblastoma cells.

    PubMed Central

    Zafra, F; Aragón, C; Giménez, C

    1994-01-01

    L-Proline transport in C6 glioblastoma cells takes place mainly via a saturable Na(+)-dependent mechanism. The uptake process can be discriminated into two components, system A and system ASC. A minor proportion of L-proline transport is carried out by the ASC system, which appears to be constitutively expressed by the cell, but most is by system A which shows adaptive responses to amino acid deprivation and sensitivity to N-methyl-alpha-aminoisobutyric acid. The transport system is inhibited by proline derivatives, such as methyl and benzyl esters, and also hydroxyproline, and is stereospecific. Incubation of glioblastoma cells with phorbol 12-myristate 13-acetate led to concentration- and time-dependent decreases in L-proline transport. This effect could be mimicked by exogenous phospholipase C. Proline transport is significantly stimulated in the presence of Ca(2+)-mobilization agents and strongly inhibited in the absence of Ca2+. The present data suggest a complex regulation of L-proline transport by different kinases in glioblastoma cells. PMID:7945191

  13. A study of characteristics of intercity transportation systems. Phase 1: Definition of transportation comparison methodology

    NASA Technical Reports Server (NTRS)

    English, J. M.; Smith, J. L.; Lifson, M. W.

    1978-01-01

    The objectives of this study are: (1) to determine a unified methodological framework for the comparison of intercity passenger and freight transportation systems; (2) to review the attributes of existing and future transportation systems for the purpose of establishing measures of comparison. These objectives were made more specific to include: (1) development of a methodology for comparing long term transportation trends arising from implementation of various R&D programs; (2) definition of value functions and attribute weightings needed for further transportation goals.

  14. Characteristics of the polymer transport in ratchet systems.

    PubMed

    Kauttonen, Janne; Merikoski, Juha

    2010-04-01

    Molecules with complex internal structure in time-dependent periodic potentials are studied by using short Rubinstein-Duke model polymers as an example. We extend our earlier work on transport in stochastically varying potentials to cover also deterministic potential switching mechanisms, energetic efficiency, and nonuniform charge distributions. We also use currents in the nonequilibrium steady state to identify the dominating mechanisms that lead to polymer transportation and analyze the evolution of the macroscopic state (e.g., total and head-to-head lengths) of the polymers. Several numerical methods are used to solve the master equations and nonlinear optimization problems. The dominating transport mechanisms are found via graph optimization methods. The results show that small changes in the molecule structure and the environment variables can lead to large increases of the drift. The drift and the coherence can be amplified by using deterministic flashing potentials and customized polymer charge distributions. Identifying the dominating transport mechanism by graph analysis tools is found to give insight in how the molecule is transported by the ratchet effect.

  15. Characteristics of endoplasmic reticulum-derived transport vesicles

    PubMed Central

    1994-01-01

    We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules. PMID:8063853

  16. Transport characteristics of tramadol in the blood-brain barrier.

    PubMed

    Kitamura, Atsushi; Higuchi, Kei; Okura, Takashi; Deguchi, Yoshiharu

    2014-10-01

    Tramadol is a centrally acting analgesic whose action is mediated by both agonistic activity at opioid receptors and inhibitory activity on neuronal reuptake of monoamines. The purpose of this study was to characterize the blood-brain barrier (BBB) transport of tramadol by means of microdialysis studies in rat brain and in vitro studies with human immortalized brain capillary endothelial cells (hCMEC/D3). The Kp,uu,brain value of tramadol determined by rat brain microdialysis was greater than unity, indicating that tramadol is actively taken up into the brain across the BBB. Tramadol was transported into hCMEC/D3 cells in a concentration-dependent manner. The uptake was inhibited by type II cations (pyrilamine, verapamil, etc.), but not by substrates of organic cation transporter OCTs or OCTN2. It was also inhibited by a metabolic inhibitor but was independent of extracellular sodium or membrane potential. The uptake was altered by changes of extracellular pH, and by ammonium chloride-induced intracellular acidification, suggesting that transport of tramadol is driven by an oppositely directed proton gradient. Thus, our in vitro and in vivo results suggest that tramadol is actively transported, at least in part, from blood to the brain across the BBB by proton-coupled organic cation antiporter.

  17. A study of characteristics of intercity transportation systems. Phase 1: Definition of transportation comparison methodology

    NASA Technical Reports Server (NTRS)

    English, J. M.; Smith, J. L.; Lifson, M. W.

    1978-01-01

    Decision making in early transportation planning must be responsive to complex value systems representing various policies and objectives. The assessment of alternative transportation concepts during the early initial phases of the system life cycle, when supportive research and technology development activities are defined, requires estimates of transportation, environmental, and socio-economic impacts throughout the system life cycle, which is a period of some 40 or 50 years. A unified methodological framework for comparing intercity passenger and freight transportation systems is described and is extended to include the comparison of long term transportation trends arising from implementation of the various R & D programs. The attributes of existing and future transportation systems are reviewed in order to establish measures for comparison, define value functions, and attribute weightings needed for comparing alternative policy actions for furthering transportation goals. Comparison criteria definitions and an illustrative example are included.

  18. Action potential initiation and propagation in CA3 pyramidal axons.

    PubMed

    Meeks, Julian P; Mennerick, Steven

    2007-05-01

    Thin, unmyelinated axons densely populate the mammalian hippocampus and cortex. However, the location and dynamics of spike initiation in thin axons remain unclear. We investigated basic properties of spike initiation and propagation in CA3 neurons of juvenile rat hippocampus. Sodium channel alpha subunit distribution and local applications of tetrodotoxin demonstrate that the site of first threshold crossing in CA3 neurons is approximately 35 microm distal to the soma, somewhat more proximal than our previous estimates. This discrepancy can be explained by the finding, obtained with simultaneous whole cell somatic and extracellular axonal recordings, that a zone of axon stretching to approximately 100 microm distal to the soma reaches a maximum rate of depolarization nearly synchronously by the influx of sodium from the high-density channels. Models of the proximal axon incorporating observed distributions of sodium channel staining recapitulated salient features of somatic and axonal spike waveforms, including the predicted initiation zone, characteristic spike latencies, and conduction velocity. The preferred initiation zone was unaltered by stimulus strength or repetitive spiking, but repetitive spiking increased threshold and significantly slowed initial segment recruitment time and conduction velocity. Our work defines the dynamics of initiation and propagation in hippocampal principal cell axons and may help reconcile recent controversies over initiation site in other axons.

  19. Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons

    NASA Technical Reports Server (NTRS)

    Kirkpatrick, L. L.; Witt, A. S.; Payne, H. R.; Shine, H. D.; Brady, S. T.

    2001-01-01

    Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.

  20. Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons

    NASA Technical Reports Server (NTRS)

    Kirkpatrick, L. L.; Witt, A. S.; Payne, H. R.; Shine, H. D.; Brady, S. T.

    2001-01-01

    Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.

  1. Transport and screen blockage characteristics of reflective metallic insulation materials

    SciTech Connect

    Brocard, D.N.

    1984-01-01

    In the event of a LOCA within a nuclear power plant, it is possible for insulation debris to be generated by the break jet. Such debris has the potential for PWR sump screen (or BWR RHR suction inlet) blockage and thus can affect the long-term recirculation capability. In addition to the variables of break jet location and orientation, the types and quantities of debris which could be generated are dependent on the insulation materials employed. This experimental investigation was limited to reflective metallic insulation and components thereof. The study was aimed at determining the flow velocities needed to transport the insulation debris to the sump screens and the resulting modes of screen blockage. The tests revealed that thin metallic foils (0.0025 in. and 0.004 in.) could transport at low flow velocities, 0.2 to 0.5 ft/sec. Thicker foils (0.008 in.) transported at higher velocities, 0.4 to 0.8 ft/sec, and as fabricated half cylinder insulation units required velocities in excess of 1.0 ft/sec for transport. The tests also provided information on screen blockage patterns that showed blockage could occur at the lower portion of the screen as foils readily flipped on the screen when reaching it.

  2. Characteristics of electron internal transport barrier in Heliotron J

    NASA Astrophysics Data System (ADS)

    Kenmochi, N.; Minami, T.; Takahashi, C.; Mochizuki, S.; Nishioka, K.; Kobayashi, S.; Nagasaki, K.; Nakamura, Y.; Okada, H.; Kado, S.; Yamamoto, S.; Ohshima, S.; Konoshima, S.; Weir, G. M.; Otani, Y.; Mizuuchi, T.

    2017-05-01

    The formation of an electron internal transport barrier (eITB) has been observed for the first time with centrally focused electron cyclotron heating (ECH) microwaves injected into plasma in Heliotron J. When the heating power per electron density ({P}{ECH}/{\\bar{n}}{{e}}) exceeds a threshold of 250× {10}-19 {kW} {{{m}}}3, transient increases of both the central T e and the core T e gradients are observed. A neoclassical (NC) calculation using the Sugama-Nishimura moment method predicts that the large positive radial electric field (E r) is formed in the core region. Heat transport analysis shows a significant reduction of the effective electron thermal diffusivity in the plasma with the eITB related to that without the eITB. The large gap between the experimentally obtained effective thermal diffusivity and the NC thermal diffusivity suggests that the suppression of anomalous transport contributes to the core improved confinement of the eITB plasma. The electron cyclotron emission measurement shows both the transient increase and the hysteresis phenomena during the eITB formation.

  3. Quantifying the Solid State Charge Transport Characteristics of Radical Polymers

    NASA Astrophysics Data System (ADS)

    Baradwaj, Aditya; Rostro, Lizbeth; Boudouris, Bryan

    2014-03-01

    Radical polymers are an emerging class of functional macromolecules that have shown immense potential to transport charge in electrolyte-supported applications. However, quantifying the ability of these non-conjugated macromolecules to conduct charge has not been as well-studied in the solid state. Here, we present the characterization of the charge transport capability of a radical polymer, poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate) (PTMA) with well-defined molecular properties in the solid state. We show that charge transport occurs across the singularly occupied molecular orbital (SOMO) level of PTMA, and that this level is 5.2 eV removed from free vacuum. Additionally, we have measured the space-charge limited hole and electron mobility values of PTMA. We find that the mobility values of these radical polymers are of the same order (10-4 cm2 V-1 s-1) of many common conjugated polymers [ e . g . , poly(3-hexylthiophene) (P3HT)]. Furthermore, because the polymer backbone is non-conjugated, these macromolecules are extremely transparent. As such, we anticipate that radical polymers could become an important component of many transparent flexible electronic applications.

  4. 78 FR 15112 - Rulemaking Advisory Committee; Transport Airplane Performance and Handling Characteristics-New Task

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-08

    ... Federal Aviation Administration Aviation Rulemaking Advisory Committee; Transport Airplane Performance and Handling Characteristics--New Task AGENCY: Federal Aviation Administration (FAA), DOT. ACTION: Notice of new task assignment for the Aviation Rulemaking Advisory Committee (ARAC). SUMMARY: The FAA assigned...

  5. Simulator study of flight characteristics of several large, dissimilar, cargo transport airplanes during approach and landing

    NASA Technical Reports Server (NTRS)

    Grantham, W. D.; Smith, P. M.; Deal, P. L.; Neely, W. R., Jr.

    1984-01-01

    A six-degree-of-freedom, ground based simulator study is conducted to evaluate the low-speed flight characteristics of four dissimilar cargo transport airplanes. These characteristics are compared with those of a large, present-day (reference) transport configuration similar to the Lockheed C-5A airplane. The four very large transport concepts evaluated consist of single-fuselage, twin-fuselage, triple-fuselage, and span-loader configurations. The primary piloting task is the approach and landing operation. The results of his study indicate that all four concepts evaluated have unsatisfactory longitudinal and lateral directional low speed flight characteristics and that considerable stability and control augmentation would be required to improve these characteristics (handling qualities) to a satisfactory level. Through the use of rate command/attitude hold augmentation in the pitch and roll axes, and the use of several turn-coordination features, the handling qualities of all four large transports simulated are improved appreciably.

  6. Kinesin accumulation in chick spinal axonal swellings with beta,beta'-iminodipropionitrile (IDPN) intoxication.

    PubMed

    Toyoshima, I; Kato, K; Sugawara, M; Wada, C; Masamune, O

    1998-06-19

    Kinesin is a major molecular motor responsible for anterograde axonal transport. Chicks were injected with beta,beta'-iminodipropionitrile (IDPN) to induce axonal swellings in spinal motor neurons and spinal sensory ganglion neurons. Cylindrical swollen axons were found in the anterior horn and anterior funiculus of the spinal cord, anterior root, and spinal ganglia. All of the axonal swellings were heavily stained with two anti-kinesin monoclonal antibodies. The swellings were mildly stained with an anti-cytoplasmic dynein and anti-tubulin antibodies, and weakly stained with an anti-tau antibody. These suggest the isolated disturbance of kinesin transport with neurofilament accumulation in IDPN intoxication.

  7. Rafting along the axon on Unc104 motors.

    PubMed

    Scholey, Jonathan M

    2002-05-01

    Neurotransmission depends upon the fast axonal transport of synaptic vesicle precursors by the monomeric kinesin Unc104, a motor whose mechanism of action is a topic of debate. New work suggests that the formation of lipid raft domains triggers the assembly of vesicle-bound Unc104 dimers and the concomitant activation of processive movement, facilitating efficient long-range vesicle transport.

  8. Modeling molecular mechanisms in the axon

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

    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.

  9. Modeling molecular mechanisms in the axon

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

    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.

  10. Axon-glial interactions at the Drosophila CNS midline.

    PubMed

    Crews, Stephen T

    2010-01-01

    The glia that reside at the midline of the Drosophila CNS are an important embryonic signaling center and also wrap the axons that cross the CNS. The development of the midline glia (MG) is characterized by migration, ensheathment, subdivision of axon commissures, apoptosis, and the extension of glial processes. All of these events are characterized by cell-cell contact between MG and adjacent neurons. Cell adhesion and signaling proteins that mediate different aspects of MG development and MG-neuron interactions have been identified. This provides a foundation for ultimately obtaining an integrated picture of how the MG assemble into a characteristic axonal support structure in the CNS.

  11. Microfluidic control of axonal guidance

    NASA Astrophysics Data System (ADS)

    Gu, Ling; Black, Bryan; Ordonez, Simon; Mondal, Argha; Jain, Ankur; Mohanty, Samarendra

    2014-10-01

    The precision of axonal pathfinding and the accurate formation of functional neural circuitry are crucial for an organism during development as well as during adult central and peripheral nerve regeneration. While chemical cues are believed to be primarily responsible for axonal pathfinding, we hypothesize that forces due to localized fluid flow may directly affect neuronal guidance during early organ development. Here, we report direct evidence of fluid flow influencing axonal migration, producing turning angles of up to 90°. Microfluidic flow simulations indicate that an axon may experience significant bending force due to cross-flow, which may contribute to the observed axonal turning. This method of flow-based guidance was successfully used to fasciculate one advancing axon onto another, showcasing the potential of this technique to be used for the formation of in vitro neuronal circuits.

  12. Minimizing the caliber of myelinated axons by means of nodal constrictions

    PubMed Central

    Johnson, Christopher; Holmes, William R.; Brown, Anthony

    2015-01-01

    In myelinated axons, most of the voltage-gated ion channels are concentrated at the nodes of Ranvier, which are short gaps in the myelin sheath. This arrangement leads to saltatory conduction and a larger conduction velocity than in nonmyelinated axons. Intriguingly, axons in the peripheral nervous system that exceed about 2 μm in diameter exhibit a characteristic narrowing of the axon at nodes that results in a local reduction of the axonal cross-sectional area. The extent of constriction increases with increasing internodal axonal caliber, reaching a threefold reduction in diameter for the largest axons. In this paper, we use computational modeling to investigate the effect of nodal constrictions on axonal conduction velocity. For a fixed number of ion channels, we find that there is an optimal extent of nodal constriction which minimizes the internodal axon caliber that is required to achieve a given target conduction velocity, and we show that this is sensitive to the precise geometry of the axon and myelin sheath in the flanking paranodal regions. Thus axonal constrictions at nodes of Ranvier appear to be a biological adaptation to minimize axonal volume, thereby maximizing the spatial and metabolic efficiency of these processes, which can be a significant evolutionary constraint. We show that the optimal nodal morphologies are relatively insensitive to changes in the number of nodal sodium channels. PMID:26224772

  13. Cellular Strategies of Axonal Pathfinding

    PubMed Central

    Raper, Jonathan; Mason, Carol

    2010-01-01

    Axons follow highly stereotyped and reproducible trajectories to their targets. In this review we address the properties of the first pioneer neurons to grow in the developing nervous system and what has been learned over the past several decades about the extracellular and cell surface substrata on which axons grow. We then discuss the types of guidance cues and their receptors that influence axon extension, what determines where cues are expressed, and how axons respond to the cues they encounter in their environment. PMID:20591992

  14. Experimental subarachnoid haemorrhage results in multifocal axonal injury.

    PubMed

    Kummer, Terrance T; Magnoni, Sandra; MacDonald, Christine L; Dikranian, Krikor; Milner, Eric; Sorrell, James; Conte, Valeria; Benetatos, Joey J; Zipfel, Gregory J; Brody, David L

    2015-09-01

    The great majority of acute brain injury results from trauma or from disorders of the cerebrovasculature, i.e. ischaemic stroke or haemorrhage. These injuries are characterized by an initial insult that triggers a cascade of injurious cellular processes. The nature of these processes in spontaneous intracranial haemorrhage is poorly understood. Subarachnoid haemorrhage, a particularly deadly form of intracranial haemorrhage, shares key pathophysiological features with traumatic brain injury including exposure to a sudden pressure pulse. Here we provide evidence that axonal injury, a signature characteristic of traumatic brain injury, is also a prominent feature of experimental subarachnoid haemorrhage. Using histological markers of membrane disruption and cytoskeletal injury validated in analyses of traumatic brain injury, we show that axonal injury also occurs following subarachnoid haemorrhage in an animal model. Consistent with the higher prevalence of global as opposed to focal deficits after subarachnoid haemorrhage and traumatic brain injury in humans, axonal injury in this model is observed in a multifocal pattern not limited to the immediate vicinity of the ruptured artery. Ultrastructural analysis further reveals characteristic axonal membrane and cytoskeletal changes similar to those associated with traumatic axonal injury. Diffusion tensor imaging, a translational imaging technique previously validated in traumatic axonal injury, from these same specimens demonstrates decrements in anisotropy that correlate with histological axonal injury and functional outcomes. These radiological indicators identify a fibre orientation-dependent gradient of axonal injury consistent with a barotraumatic mechanism. Although traumatic and haemorrhagic acute brain injury are generally considered separately, these data suggest that a signature pathology of traumatic brain injury-axonal injury-is also a functionally significant feature of subarachnoid haemorrhage, raising

  15. Experimental subarachnoid haemorrhage results in multifocal axonal injury

    PubMed Central

    Magnoni, Sandra; MacDonald, Christine L.; Dikranian, Krikor; Milner, Eric; Sorrell, James; Conte, Valeria; Benetatos, Joey J.; Zipfel, Gregory J.; Brody, David L.

    2015-01-01

    The great majority of acute brain injury results from trauma or from disorders of the cerebrovasculature, i.e. ischaemic stroke or haemorrhage. These injuries are characterized by an initial insult that triggers a cascade of injurious cellular processes. The nature of these processes in spontaneous intracranial haemorrhage is poorly understood. Subarachnoid haemorrhage, a particularly deadly form of intracranial haemorrhage, shares key pathophysiological features with traumatic brain injury including exposure to a sudden pressure pulse. Here we provide evidence that axonal injury, a signature characteristic of traumatic brain injury, is also a prominent feature of experimental subarachnoid haemorrhage. Using histological markers of membrane disruption and cytoskeletal injury validated in analyses of traumatic brain injury, we show that axonal injury also occurs following subarachnoid haemorrhage in an animal model. Consistent with the higher prevalence of global as opposed to focal deficits after subarachnoid haemorrhage and traumatic brain injury in humans, axonal injury in this model is observed in a multifocal pattern not limited to the immediate vicinity of the ruptured artery. Ultrastructural analysis further reveals characteristic axonal membrane and cytoskeletal changes similar to those associated with traumatic axonal injury. Diffusion tensor imaging, a translational imaging technique previously validated in traumatic axonal injury, from these same specimens demonstrates decrements in anisotropy that correlate with histological axonal injury and functional outcomes. These radiological indicators identify a fibre orientation-dependent gradient of axonal injury consistent with a barotraumatic mechanism. Although traumatic and haemorrhagic acute brain injury are generally considered separately, these data suggest that a signature pathology of traumatic brain injury—axonal injury—is also a functionally significant feature of subarachnoid haemorrhage

  16. Electronic transport characteristics in silicon nanotube field-effect transistors

    NASA Astrophysics Data System (ADS)

    Shan, Guangcun; Wang, Yu; Huang, Wei

    2011-07-01

    The successful synthesis of silicon nanotubes (SiNTs) has been reported, making these nanostructures a new novel candidate for future nanodevices. By self-consistently solving the Poisson equations using the non-equilibrium Green's function (NEGF) formalism, we investigate the electronic transport and the role of gate bias in affecting the drive current of single-walled silicon nanotube (SW-SiNT) field-effect transistors (FETs). By comparison of a SW-CNT FET, it is found that the SW-SiNT with a high- k HfO gate oxide is a promising candidate for nanotube transistor with better performance. The results discussed here would serve as a versatile and powerful guideline for future experimental studies of SW-SiNT-based transistor with the purpose of exploring device application for nanoelectronics.

  17. Imp promotes axonal remodeling by regulating profilin mRNA during brain development.

    PubMed

    Medioni, Caroline; Ramialison, Mirana; Ephrussi, Anne; Besse, Florence

    2014-03-31

    Neuronal remodeling is essential for the refinement of neuronal circuits in response to developmental cues [1-4]. Although this process involves pruning or retraction of axonal projections followed by axonal regrowth and branching, how these steps are controlled is poorly understood. Drosophila mushroom body (MB) γ neurons provide a paradigm for the study of neuronal remodeling, as their larval axonal branches are pruned during metamorphosis and re-extend to form adult-specific branches [5]. Here, we identify the RNA binding protein Imp as a key regulator of axonal remodeling. Imp is the sole fly member of a conserved family of proteins that bind target mRNAs to promote their subcellular targeting [6-12]. We show that whereas Imp is dispensable for the initial growth of MB γ neuron axons, it is required for the regrowth and ramification of axonal branches that have undergone pruning. Furthermore, Imp is actively transported to axons undergoing developmental remodeling. Finally, we demonstrate that profilin mRNA is a direct and functional target of Imp that localizes to axons and controls axonal regrowth. Our study reveals that mRNA localization machineries are actively recruited to axons upon remodeling and suggests a role of mRNA transport in developmentally programmed rewiring of neuronal circuits during brain maturation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Turbulence characteristics in grassland canopies and implications for tracer transport

    NASA Astrophysics Data System (ADS)

    Nemitz, E.; Loubet, B.; Lehmann, B. E.; Cellier, P.; Neftel, A.; Jones, S. K.; Hensen, A.; Ihly, B.; Tarakanov, S. V.; Sutton, M. A.

    2009-08-01

    In-canopy turbulence is a required input to study pollutant cycling and chemistry within plant canopies and to link concentrations and sources. Despite the importance of grasslands worldwide, most previous work has focused on forests and crops. Here, turbulence parameters in a mature agricultural grassland canopy were measured with a combination of a small ultrasonic anemometer, hotwire anemometry and a radon (Rn) tracer technique, as part of a measurement to study ammonia (NH3) exchange with grassland. The measurements are used to derive vertical profiles of basic turbulent parameters, for quadrant-hole analysis of the two-parametric frequency distributions of u'-w' and to derive in-canopy eddy diffusivities as input for models of in-canopy tracer transport. The results are in line with previous measurements on taller canopies, but shows increased decoupling between in-canopy flow and above-canopy turbulence. The comparison of sonic anemometry and Rn measurements implies that Lagrangian time-scales must decrease sharply at the ground, with important implications for estimating the magnitude of ground-level and soil emissions from concentration measurements. Atmospheric stability above and within the canopy has little influence on the standard deviation of vertical wind component inside the canopy. Use of the turbulence parameters in an analytical Lagrangian framework, which is here validated for heat transfer, suggests that measured in-canopy profiles of NH3 are consistent with a ground-level source, presumably from senescent plant parts, which is recaptured by the overlying canopy.

  19. Turbulence characteristics in grassland canopies and implications for tracer transport

    NASA Astrophysics Data System (ADS)

    Nemitz, E.; Loubet, B.; Lehmann, B. E.; Cellier, P.; Neftel, A.; Jones, S. K.; Hensen, A.; Ihly, B.; Tarakanov, S. V.; Sutton, M. A.

    2009-01-01

    In-canopy turbulence is a required input to study pollutant cycling and chemistry within plant canopies and to link concentrations and sources. Despite the importance of grasslands worldwide, most previous work has focused on forests and crops. Here, turbulence parameters in a mature agricultural grassland canopy were measured with a combination of a small ultrasonic anemometer, hotwire anemometry and a radon (Rn) tracer technique, as part of a measurement to study ammonia (NH3) exchange with grassland. The measurements are used to derive vertical profiles of basic turbulent parameters, for quadrant-hole analysis of the two-parametric frequency distributions of u'-w' and to derive in-canopy eddy diffusivities as input for models of in-canopy tracer transport. The results are in line with previous measurements on taller canopies, but shows increased decoupling between in-canopy flow and above-canopy turbulence. The comparison of sonic anemometry and Rn measurements implies that Lagrangian time-scales must decrease sharply at the ground, with important implications for estimating the magnitude of ground-level and soil emissions from concentration measurements. Atmospheric stability above and within the canopy has little influence on the standard deviation of vertical wind component inside the canopy. Use of the turbulence parameters in an analytical Lagrangian framework, which is here validated for heat transfer, suggests that measured in-canopy profiles of NH3 are consistent with a ground-level source, presumably from senescent plant parts, which is recaptured by the overlying canopy.

  20. Seismic reflection characteristics of naturally-induced subsidence affecting transportation

    USGS Publications Warehouse

    Miller, R.D.; Xia, J.; Steeples, D.W.

    2009-01-01

    High-resolution seismic reflections have been used effectively to investigate sinkholes formed from the dissolution of a bedded salt unit found throughout most of Central Kansas. Surface subsidence can have devastating effects on transportation structures. Roads, rails, bridges, and pipelines can even be dramatically affected by minor ground instability. Areas susceptible to surface subsidence can put public safety at risk. Subsurface expressions significantly larger than surface depressions are consistently observed on seismic images recorded over sinkholes in Kansas. Until subsidence reaches the ground surface, failure appears to be controlled by compressional forces evidenced by faults with reverse orientation. Once a surface depression forms or dissolution of the salt slows or stops, subsidence structures are consistent with a tensional stress environment with prevalent normal faults. Detecting areas of rapid subsidence potential, prior to surface failure, is the ultimate goal of any geotechnical survey where the ground surface is susceptible to settling. Seismic reflection images have helped correlate active subsidence to dormant paleofeatures, project horizontal growth of active sinkholes based on subsurface structures, and appraise the risk of catastrophic failure. ?? China University of Geosciences (Wuhan) and Springer-Verlag GmbH 2009.

  1. Recording Large Extracellular Spikes in Microchannels along Many Axonal Sites from Individual Neurons

    PubMed Central

    Lewandowska, Marta K.; Bakkum, Douglas J.; Rompani, Santiago B.; Hierlemann, Andreas

    2015-01-01

    The numerous connections between neuronal cell bodies, made by their dendrites and axons, are vital for information processing in the brain. While dendrites and synapses have been extensively studied, axons have remained elusive to a large extent. We present a novel platform to study axonal physiology and information processing based on combining an 11,011-electrode high-density complementary metal-oxide semiconductor microelectrode array with a poly(dimethylsiloxane) channel device, which isolates axons from somas and, importantly, significantly amplifies recorded axonal signals. The combination of the microelectrode array with recording and stimulation capability with the microfluidic isolation channels permitted us to study axonal signal behavior at great detail. The device, featuring two culture chambers with over 30 channels spanning in between, enabled long-term recording of single spikes from isolated axons with signal amplitudes of 100 μV up to 2 mV. Propagating signals along axons could be recorded with 10 to 50 electrodes per channel. We (i) describe the performance and capabilities of our device for axonal electrophysiology, and (ii) present novel data on axonal signals facilitated by the device. Spontaneous action potentials with characteristic shapes propagated from somas along axons between the two compartments, and these unique shapes could be used to identify individual axons within channels that contained many axonal branches. Stimulation through the electrode array facilitated the identification of somas and their respective axons, enabling interfacing with different compartments of a single cell. Complex spike shapes observed in channels were traced back to single cells, and we show that more complicated spike shapes originate from a linear superposition of multiple axonal signals rather than signal distortion by the channels. PMID:25734567

  2. Characteristics of mammalian Rh glycoproteins (SLC42 transporters) and their role in acid-base transport.

    PubMed

    Nakhoul, Nazih L; Lee Hamm, L

    2013-01-01

    The mammalian Rh glycoproteins belong to the solute transporter family SLC42 and include RhAG, present in red blood cells, and two non-erythroid members RhBG and RhCG that are expressed in various tissues, including kidney, liver, skin and the GI tract. The Rh proteins in the red blood cell form an "Rh complex" made up of one D-subunit, one CE-subunit and two RhAG subunits. The Rh complex has a well-known antigenic effect but also contributes to the stability of the red cell membrane. RhBG and RhCG are related to the NH4(+) transporters of the yeast and bacteria but their exact function is yet to be determined. This review describes the expression and molecular properties of these membrane proteins and their potential role as NH3/NH4(+) and CO2 transporters. The likelihood that these proteins transport gases such as CO2 or NH3 is novel and significant. The review also describes the physiological importance of these proteins and their relevance to human disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Characteristics of Mammalian Rh Glycoproteins (SLC42 transporters) and Their Role in Acid-Base Transport

    PubMed Central

    Nakhoul, Nazih L.; Hamm, L. Lee

    2012-01-01

    The mammalian Rh glycoproteins belong to the solute transporter family SLC42 and include RhAG, present in red blood cells, and two non-erythroid members RhBG and RhCG that are expressed in various tissues, including kidney, liver, skin and the GI tract. The Rh proteins in the red blood cell form an “Rh complex” made up of one D-subunit, one CE-subunit and two RhAG subunits. The Rh complex has a well-known antigenic effect but also contributes to the stability of the red cell membrane. RhBG and RhCG are related to the NH4+ transporters of the yeast and bacteria but their exact function is yet to be determined. This review describes the expression and molecular properties of these membrane proteins and their potential role as NH3/NH4+ and CO2 transporters. The likelihood that these proteins transport gases such as CO2 or NH3 is novel and significant. The review also describes the physiological importance of these proteins and their relevance to human disease. PMID:23506896

  4. Transport and subcellular distribution characteristics of human erythrocyte glucose transporter fused in adipocytes

    SciTech Connect

    Jo, Inho.

    1990-01-01

    Purified human erythrocyte glucose transporters (HEGT) were incorporated into the rat epididymal adipocytes by polyethylene glycol (PEG)-induced fusion. The incorporation of HEGT was found to be dependent on both the molecular weight and the concentration of PEG. Optimal incorporation of HEGT occurred when 10% PEG 8000 was used. This incorporation was found to be proportional to the increasing amounts of HEGT used. Morphology tests showed very little surface adsorption of HEGT on adipocytes after fusion. Transport activity of fused HEGT was studied by measuring equilibrium exchange of 3-O-methylglucose. In adipocytes employed this fusion protocols, the transport rate increased significantly compared with cells treated under non-fusion conditions. This fold increase was directly proportional to the amount of HEGT recovered in the plasma membrane (PM). Furthermore, calculated turnover number of HEGT in fused adipocytes was as high as that of the native adipocyte glucose transporter (AGT). Subcellular distribution of HEGT in fused adipocytes was assessed using ({sup 3}H)cytochalasin B-labeled HEGT vesicles. HEGT was distributed in each subcellular organelle at specific ratios. This relative distribution was almost constant regardless of the amount of HEGT used. The distributions of lipid-labeled HEGT, fluid-phase endocytosed HEGT and free HEGT mixed to adipocyte homogenate were shown to be completely different from that of protein-labeled HEGT fusion.

  5. The Geochemical Behavior and Transport Characteristics of Xenoestrogens

    NASA Astrophysics Data System (ADS)

    Wallace, T. C.; Bennett, P. C.

    2003-12-01

    Xenoestrogens are estrogenic active synthetic chemicals that mimic the actions of female sex hormones. Xenoestrogens can be produced synthetically and naturally, and exposure can occur from a variety of sources- food additives, plastics, pesticides, or pharmaceuticals. These environmental chemicals are also known as endocrine disruptors because exposure to low doses (ng/L) have been linked to adverse effects in the reproductive and developmental stages in aquatic species (i.e. reproductive anomalies, feminization, infertility, alterations in growth during life cycles, and changes in community structures). Determining the exposure risks of these toxicological compounds, however, requires a better understanding of the geochemical behavior and transport of synthetic estrogens it is discharged to. Estrogen and its metabolites are also useful tracers because of their specific medical usage (sources from birth control pills, estrogen replacement therapy, and livestock farming), slow degradation before excretion, and unique physiochemical properties (low volatility, hydrophobicity, and high Kow). Estradiol concentrations analyzed by an enzyme-linked immunoassay (ELISA) show that <2-55 ng/L are discharged to Walnut Creek, a stream that also connects to the Colorado River(TX). The bioavailability of these compounds is affected by sorption processes, where xenoestrogens become associated with solid phases. A series of batch sorption experiments using sediment collected from Walnut Creek downstream of an Austin waste water treatment plant and synthetic estrogen standards (Simga@ Estrone, 17B-Estradiol, and Estriol), examined the distribution of estrogen between solid and aqueous phases. Analysis of the concentrations sorbed to sediment result in Freundlich sorption isotherms using HPLC/UV techniques (High Performance Liquid Chromatography with UV detectors- 220 and 280nm). Sorption occurs rapidly with 98% of 17B-Estradiol sorbed within 30 minutes (Estriol=80%, Estrone=95

  6. Axon density and axon orientation dispersion in children born preterm.

    PubMed

    Kelly, Claire E; Thompson, Deanne K; Chen, Jian; Leemans, Alexander; Adamson, Christopher L; Inder, Terrie E; Cheong, Jeanie L Y; Doyle, Lex W; Anderson, Peter J

    2016-09-01

    Very preterm birth (VPT, <32 weeks' gestation) is associated with altered white matter fractional anisotropy (FA), the biological basis of which is uncertain but may relate to changes in axon density and/or dispersion, which can be measured using Neurite Orientation Dispersion and Density Imaging (NODDI). This study aimed to compare whole brain white matter FA, axon dispersion, and axon density between VPT children and controls (born ≥37 weeks' gestation), and to investigate associations with perinatal factors and neurodevelopmental outcomes. FA, neurite dispersion, and neurite density were estimated from multishell diffusion magnetic resonance images for 145 VPT and 33 control 7-year-olds. Diffusion values were compared between groups and correlated with perinatal factors (gestational age, birthweight, and neonatal brain abnormalities) and neurodevelopmental outcomes (IQ, motor, academic, and behavioral outcomes) using Tract-Based Spatial Statistics. Compared with controls, VPT children had lower FA and higher axon dispersion within many major white matter fiber tracts. Neonatal brain abnormalities predicted lower FA and higher axon dispersion in many major tracts in VPT children. Lower FA, higher axon dispersion, and lower axon density in various tracts correlated with poorer neurodevelopmental outcomes in VPT children. FA and NODDI measures distinguished VPT children from controls and were associated with neonatal brain abnormalities and neurodevelopmental outcomes. This study provides a more detailed and biologically meaningful interpretation of white matter microstructure changes associated with prematurity. Hum Brain Mapp 37:3080-3102, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Motile axonal mitochondria contribute to the variability of presynaptic strength.

    PubMed

    Sun, Tao; Qiao, Haifa; Pan, Ping-Yue; Chen, Yanmin; Sheng, Zu-Hang

    2013-08-15

    One of the most notable characteristics of synaptic transmission is the wide variation in synaptic strength in response to identical stimulation. In hippocampal neurons, approximately one-third of axonal mitochondria are highly motile, and some dynamically pass through presynaptic boutons. This raises a fundamental question: can motile mitochondria contribute to the pulse-to-pulse variability of presynaptic strength? Recently, we identified syntaphilin as an axonal mitochondrial-docking protein. Using hippocampal neurons and slices of syntaphilin knockout mice, we demonstrate that the motility of axonal mitochondria correlates with presynaptic variability. Enhancing mitochondrial motility increases the pulse-to-pulse variability, whereas immobilizing mitochondria reduces the variability. By dual-color live imaging at single-bouton levels, we further show that motile mitochondria passing through boutons dynamically influence synaptic vesicle release, mainly by altering ATP homeostasis in axons. Thus, our study provides insight into the fundamental properties of the CNS to ensure the plasticity and reliability of synaptic transmission.

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

    PubMed

    Sepehrband, Farshid; Alexander, Daniel C; Clark, Kristi A; Kurniawan, Nyoman D; Yang, Zhengyi; Reutens, David C

    2016-01-01

    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.

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

    PubMed Central

    Sepehrband, Farshid; Alexander, Daniel C.; Clark, Kristi A.; Kurniawan, Nyoman D.; Yang, Zhengyi; Reutens, David C.

    2016-01-01

    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. PMID:27303273

  10. Analysis and classification of bedload transport events with variable process characteristics

    NASA Astrophysics Data System (ADS)

    Kreisler, Andrea; Moser, Markus; Aigner, Johann; Rindler, Rolf; Tritthart, Michael; Habersack, Helmut

    2017-08-01

    Knowledge about the magnitude of bedload fluxes at given hydraulic conditions in natural streams is essential for improved process understanding, for the application, calibration and validation of bedload transport formulas, and for numerical sediment transport models. Nonetheless, extensive field measurements of bedload transport are challenging and therefore data from such efforts are rare. Bedload transport has been measured comprehensively at the downstream section of the Urslau torrent in Salzburg, Austria, since 2011. We used an integrative monitoring system that combines direct (mobile basket sampler, slot sampler) and indirect measuring devices (geophone plates). Continuous information about the intensity and distribution of bedload transport within the channel cross-section is available in high spatial and temporal resolution. Seven geophone plates at a stream width of 8 m are part of a measurement system that delivers data in 1-min intervals. These geophone data are calibrated using results of direct bedload measurements, providing an opportunity to calculate bedload rates and bedload yields in selected time periods. Continuous data on the bedload transport process over three years enabled assessing several bedload transport events. The investigation of bedload transport rate/discharge relationships reveals order-of-magnitude changes. For individual events, we observed shifts in the data, reflecting different bedload rates at comparable hydraulic conditions. This study reveals that variable sediment supply conditions affect the prevailing bedload transport rates at the Urslau stream. Calculating the bedload transport efficiency enables comparing bedload transport events that exhibit similar process characteristics. Finally, we provide a conceptual model of bedload transport process types as a function of bedload transport efficiency and dimensionless stream power.

  11. Characteristics of intermittent mitochondrial transport in guinea pig enteric nerve fibers.

    PubMed

    Vanden Berghe, Pieter; Hennig, Grant W; Smith, Terence K

    2004-04-01

    Enteric neurons controlling various gut functions are prone to oxidative insults that might damage mitochondria (e.g., intestinal inflammation). To resume local energy supply, mitochondria need to be transported. We used MitoTracker dyes and confocal microscopy to investigate basic characteristics of mitochondrial transport in guinea pig myenteric neurites. During a 10-s observation of 1 mm nerve fiber, on average, three mitochondria were transported at an average speed of 0.41 +/- 0.02 microm/s. Movement patterns were clearly erratic, and velocities were independent of mitochondrial size. The velocity oscillated periodically ( approximately 6 s) but was not consistently affected by structures such as en route boutons, bifurcations, or stationary mitochondria. Also, mitochondria transported in opposite directions did not necessarily affect each others' mobility. Transport was blocked by microtubule disruption (100 microM colchicine), and destabilization (1 microM cytochalasin-D) or stabilization (10 microM phalloidin) of actin filaments, respectively, decreased (0.22 +/- 0.02 microm/s, P < 0.05) or increased (0.53 +/- 0.02 microm/s, P < 0.05) transport speed. Transport was inhibited by TTX (1 microM), and removal of extracellular Ca(2+) (plus 2 mM EGTA) had no effect. However, depletion of intracellular stores (thapsigargin) reduced (to 33%) and slowed the transport significantly (0.18 +/- 0.02 microm/s, P < 0.05), suggesting an important role for stored Ca(2+) in mitochondrial transport. Transport was also reduced (to 21%) by the mitochondrial uncoupler FCCP (1 microM) in a time-dependent fashion and slowed by oligomycin (10 microM). We conclude that mitochondrial transport is remarkably independent of structural nerve fiber properties. We also show that mitochondrial transport is TTX sensitive and speeds up by stabilizing actin and that functional Ca(2+) stores are required for efficient transport.

  12. Juvenile neuroaxonal dystrophy in a Rottweiler: accumulation of synaptic proteins in dystrophic axons.

    PubMed

    Sisó, S; Ferrer, I; Pumarola, M

    2001-11-01

    Dystrophic axons in a 2-year-old male Rottweiler with neuroaxonal dystrophy have shown synaptophysin, synapsin-I, synaptosomal-associated protein of 25 kDa (SNAP-25), Rab 3a, and alpha-synuclein immunoreactivity. Similar findings have been observed in isolated dystrophic axons in the nuclei gracillis and cunneatus in five dogs aged between 14 and 18 years. Abnormal expression of integral synaptic vesicle, synaptic vesicle-associated presynaptic plasma membrane and cytosolic proteins, which participate in the trafficking, docking and fusion of the synaptic vesicle to the plasma membrane, suggest severe disruption of axonal transport in dystrophic axons in canine neuroaxonal dystrophy.

  13. Differential compartmentalization of mRNAs in squid giant axon.

    PubMed

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

    1996-11-01

    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.

  14. Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons.

    PubMed

    Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E

    2015-01-01

    The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.

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

    PubMed Central

    Twiss, Jeffery L.; Kalinski, Ashley L.; Sachdeva, Rahul; Houle, John D.

    2016-01-01

    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. PMID:27857722

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

    PubMed

    Twiss, Jeffery L; Kalinski, Ashley L; Sachdeva, Rahul; Houle, John D

    2016-09-01

    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.

  17. Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon

    PubMed Central

    Ma, Marek

    2013-01-01

    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. PMID:23969238

  18. Loss of MEC-17 Leads to Microtubule Instability and Axonal Degeneration

    PubMed Central

    Neumann, Brent; Hilliard, Massimo A.

    2014-01-01

    SUMMARY Axonal degeneration arises as a consequence of neuronal injury and is a common hallmark of a number of neurodegenerative diseases. However, the genetic causes and the cellular mechanisms that trigger this process are still largely unknown. Based on forward genetic screening in C. elegans, we have identified the α-tubulin acetyltransferase gene mec-17 as causing spontaneous, adult-onset, and progressive axonal degeneration. Loss of MEC-17 leads to microtubule instability, a reduction in mitochondrial number, and disrupted axonal transport, with altered distribution of both mitochondria and synaptic components. Furthermore, mec-17-mediated axonal degeneration occurs independently from its acetyltransferase domain; is enhanced by mutation of coel-1, a tubulin-associated molecule; and correlates with the animal’s body length. This study therefore identifies a critical role for the conserved microtubule-associated protein MEC-17 in preserving axon integrity and preventing axonal degeneration. PMID:24373971

  19. Some characteristics of membrane Cd²+ transport in rat thymocytes: an analysis using Fluo-3.

    PubMed

    Kawanai, Takuya; Fujinaga, Masahiro; Koizumi, Kazuki; Kurotani, Isao; Hashimoto, Erika; Satoh, Masaya; Imai, Shoji; Miyoshi, Norikazu; Oyama, Yasuo

    2011-10-01

    Although cadmium-induced apoptosis of lymphocytes is one of common features in the immunotoxicity of cadmium, the membrane pathway for intracellular cadmium accumulation is not fully elucidated. To characterize membrane Cd(2+) transport of rat thymocytes, the change in intracellular Cd(2+) concentration under various conditions was examined by the use of Fluo-3, a fluorescent probe for monitoring the change in intracellular concentration of divalent metal cations. The membrane Cd(2+) transport was estimated by the augmentation of Fluo-3 fluorescence induced by bath application of CdCl(2). Lowering temperature strongly suppressed the augmentation of Fluo-3 fluorescence by CdCl(2), suggesting that the metabolic process can be involved in membrane Cd(2+) transport. External acidification (decreasing pH) and membrane depolarization by adding KCl attenuated the augmentation, indicating the requirement of electrochemical driving force for membrane Cd(2+) transport into the cells. Bath application of CaCl(2) and ZnCl(2) equally decreased the augmentation, suggesting their competition with Cd(2+) at the membrane transport. The augmentation by CdCl(2) was lesser in the cells treated with N-ethylmaleimide inducing chemical depletion of cellular thiols. The result suggests the contribution of sulfhydryl groups to membrane Cd(2+) transport. Taken together, it is suggested that the cells possess a temperature-sensitive membrane Cd(2+) pathway, driven by electrochemical gradient of Cd(2+) and transmembrane potential, with competitive binding site. Based on the characteristics described above, it is unlikely that the membrane Cd(2+) transport in rat thymocytes is attributed to a single transport system although it has characteristics that are similar to those of divalent cation transporter 1.

  20. Neurofilaments form a Highly Stable Stationary Cytoskeleton After Reaching a Critical Level in Axons

    PubMed Central

    Yuan, Aidong; Sasaki, Takahiro; Rao, Mala V.; Kumar, Asok; Kanumuri, Vivek; Dunlop, David S.; Liem, Ronald K.; Nixon, Ralph A.

    2009-01-01

    The ultrastructural view of the axonal cytoskeleton as an extensively crosslinked network of neurofilaments (NFs) and other cytoskeletal polymers contrasts with the dynamic view suggested by axonal transport studies on cytoskeletal elements. Here we reconcile these perspectives by showing that neurons form a large NF network along axons which is unequivocally stationary, metabolically stable, and maintained by NFs and non-filamentous subunit assemblies undergoing slow transport by intermittent rapid movements and pauses. In mouse primary cortical neurons transfected with EGFP-NFL, formation of this stationary NF network requires a critical level of NFs, which explains its absence in NF-poor developing neurons studied previously. Most NFs at proximal axon regions were in a stationary structure coexisting with a smaller pool of moving EGFP-NFL assemblies that were mainly non-filamentous. Distally along the same axon, EGFP-labeled NFL was much less abundant and we detected only short filaments moving bidirectionally by slow transport (rapid movements and pauses) as previously described. In living mice, >25% of radiolabeled newly synthesized NFs remained in optic axons after slowly transport NFs had exited. Retained NF remained fixed over several months in a non-uniform distribution and exhibited exceptionally slow turnover (t 1/2 > 2.5 months), implying that, at steady state, >90% of NFs in mature optic axons comprise the stationary cytoskeleton and <10% are undergoing slow transport. These findings reconcile in vitro and in vivo axonal transport observations, showing that slowly transport NFs or subunit oligomers are precursors to a highly stable stationary cytoskeletal network that supports mature axons. PMID:19741138

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

    PubMed

    Mollá, Belén; Muñoz-Lasso, Diana C; Riveiro, Fátima; Bolinches-Amorós, Arantxa; Pallardó, Federico V; Fernandez-Vilata, Angel; de la Iglesia-Vaya, María; Palau, Francesc; Gonzalez-Cabo, Pilar

    2017-01-01

    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 Ca(2+) 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 Ca(2+) with Ca(2+) chelators or metalloprotease inhibitors, preventing Ca(2+)-mediated axonal injury. Thus, the modulation of Ca(2+) levels may be a relevant therapeutic target to develop early axonal protection and prevent dying-back neurodegeneration.

  2. Axonal localization of transgene mRNA in mature PNS and CNS neurons

    PubMed Central

    Willis, Dianna E.; Xu, Mei; Donnelly, Christopher J.; Tep, Chhavy; Kendall, Marvin; Erenstheyn, Marina; English, Arthur; Schanen, N. Carolyn; Kirn-Safran, Catherine B.; Yoon, Sung Ok; Bassell, Gary J.; Twiss, Jeffery L.

    2011-01-01

    Axonal mRNA transport is robust in cultured neurons but there has been limited evidence for this in vivo. We have used a genetic approach to test for in vivo axonal transport of reporter mRNAs. We show that β-actin’s 3’UTR can drive axonal localization of GFP mRNA in mature DRG neurons, but mice with γ-actin’s 3’UTR show no axonal GFP mRNA. Peripheral axotomy triggers transport of the β-actin 3’UTR containing transgene mRNA into axons. This GFP-3’β-actin mRNA accumulates in injured PNS axons before activation of the transgene promoter peaks in the DRG. Spinal cord injury also increases axonal GFP signals in mice carrying this transgene without any increase in transgene expression in the DRGs. These data show for the first time that the β-actin 3’UTR is sufficient for axonal localization in both PNS and CNS neurons in vivo. PMID:21994364

  3. Water Transport Characteristics of Gas Diffusion Layer in a PEM Fuel Cell

    SciTech Connect

    Damle, Ashok S; Cole, J Vernon

    2008-12-01

    A presentation addressing the following: Water transport in PEM Fuel Cells - a DoE Project 1. Gas Diffusion Layer--Role and Characteristics 2. Capillary Pressure Determinations of GDL Media 3. Gas Permeability Measurements of GDL Media 4. Conclusions and Future Activities

  4. Acceptability of Alternatives to Traditional Emergency Care: Patient Characteristics, Alternate Transport Modes, and Alternate Destinations.

    PubMed

    Jones, Courtney Marie Cora; Wasserman, Erin B; Li, Timmy; Shah, Manish N

    2015-01-01

    To determine the acceptability of alternatives to traditional emergency care, we assessed the proportion of subjects willing to consider alternative modes of transportation and alternative destinations. We further identified patient characteristics associated with willingness to consider these alternatives. We conducted a cross-sectional survey study in the emergency department (ED) of an academic medical center. Research assistants screened all non-critically ill ED patients for eligibility and willingness to participate and administered an interview-based survey that included questions on demographic and clinical characteristics, perceived illness severity, and acceptability of alternatives to traditional emergency care for acute illness and injuries. We calculated the proportions and 95% confidence intervals for subjects who found alternative transport modes and destinations acceptable and developed a log-binomial regression model to identify patient characteristics associated with acceptability of alternative modes of transport and alternative destinations. Complete data were available on 1,058 subjects. Forty-two percent of the study sample arrived to the ED via emergency medical services (EMS). Over two-thirds of the study sample (68.2%) was willing to consider transport via either taxi or medical transport van and 69.0% was willing to consider either transportation to an urgent care center or their primary care physician's office. Other alternatives, including delayed EMS response time, were less frequently endorsed as acceptable alternatives. Subject characteristics associated with willingness to accept alternative modes of transportation included younger age, chief complaint, previous ED use, and place of residence (p < 0.05). Subject characteristics associated with willingness to accept alternative destinations included younger age, non-white race, lower patient acuity, and lower self-perceived illness severity (p < 0.05). In our ED, some patients found

  5. Understanding electrical-thermal transport characteristics of organic semiconductors: Violation of Wiedemann-Franz law

    NASA Astrophysics Data System (ADS)

    Lu, Nianduan; Li, Ling; Gao, Nan; Liu, Ming

    2016-11-01

    Organic semiconductors exhibit plenty of attractive properties for use as thermoelectric elements. A comprehensive understanding for the electrical-thermal transport characteristics is crucial to design and fabricate the thermoelectric device. We proposed a theoretical model to investigate the electrical conductivity and the electronic thermal conductivity of organic semiconductors based on the hopping transport mechanism. The electrical-thermal transport characteristics of organic semiconductors have been analyzed in detail and compared with the experimental results and Monte Carlo simulation. The Wiedemann-Franz law, connecting the electronic thermal conductivity to the electrical conductivity of organic semiconductors, is generally found to be strongly violated under the effect of temperature, carrier concentration, energetic disorder and electric field.

  6. Gate-dependent asymmetric transport characteristics in pentacene barristors with graphene electrodes

    NASA Astrophysics Data System (ADS)

    Hwang, Wang-Taek; Min, Misook; Jeong, Hyunhak; Kim, Dongku; Jang, Jingon; Yoo, Daekyung; Jang, Yeonsik; Kim, Jun-Woo; Yoon, Jiyoung; Chung, Seungjun; Yi, Gyu-Chul; Lee, Hyoyoung; Wang, Gunuk; Lee, Takhee

    2016-11-01

    We investigated the electrical characteristics and the charge transport mechanism of pentacene vertical hetero-structures with graphene electrodes. The devices are composed of vertical stacks of silicon, silicon dioxide, graphene, pentacene, and gold. These vertical heterojunctions exhibited distinct transport characteristics depending on the applied bias direction, which originates from different electrode contacts (graphene and gold contacts) to the pentacene layer. These asymmetric contacts cause a current rectification and current modulation induced by the gate field-dependent bias direction. We observed a change in the charge injection barrier during variable-temperature current-voltage characterization, and we also observed that two distinct charge transport channels (thermionic emission and Poole-Frenkel effect) worked in the junctions, which was dependent on the bias magnitude.

  7. Transport characteristics of nanoparticle-based ferrofluids in a gel model of the brain

    PubMed Central

    Basak, Soubir; Brogan, David; Dietrich, Hans; Ritter, Rogers; Dacey, Ralph G; Biswas, Pratim

    2009-01-01

    A current advance in nanotechnology is the selective targeting of therapeutics by external magnetic field-guided delivery. This is an important area of research in medicine. The use of magnetic forces results in the formation of agglomerated structures in the field region. The transport characteristics of these agglomerated structures are explored. A nonintrusive method based on in situ light-scattering techniques is used to characterize the velocity of such particles in a magnetic field gradient. A transport model for the chain-like agglomerates is developed based on these experimental observations. The transport characteristics of magnetic nanoparticle drug carriers are then explored in gel-based simulated models of the brain. Results of such measurements demonstrate decreased diffusion of magnetic nanoparticles when placed in a high magnetic field gradient. PMID:19421367

  8. Axonal dysfunction in internal capsule is closely associated with early motor deficits after intracerebral hemorrhage in mice.

    PubMed

    Hijioka, Masanori; Anan, Junpei; Matsushita, Hideaki; Ishibashi, Hayato; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Katsuki, Hiroshi

    2016-05-01

    Previously we showed that expansion of intracerebral hemorrhage (ICH) into the internal capsule greatly aggravated neurological symptoms in mice. Here we examined ICH-associated events in the internal capsule with relation to neurological dysfunction. Corticospinal axons labeled by biotinylated dextran amine exhibited fragmented appearance after ICH induced by local injection of collagenase into the internal capsule. Fragmentation of axonal structures was confirmed by neurofilament-H immunostaining, which was evident from 6h after induction of ICH. We also observed accumulation of amyloid precursor protein, which indicated compromised axonal transport, from 3h after induction of ICH. The early defect in axonal transport was accompanied by a robust decline in motor performance. Local application of an axonal transport inhibitor colchicine to the internal capsule induced a prompt decline in motor performance, suggesting that compromised axonal transport is closely associated with early neurological dysfunction in ICH. Arrest of axonal transport and fragmentation of axonal structures were also induced by local injection of thrombin, but not by thrombin receptor activator peptide-6, a protease-activated receptor-1 agonist. These results suggest that receptor-independent actions of thrombin mediate disruption of structure and function of axons by hemorrhage expansion into the internal capsule, which leads to severe neurological dysfunction.

  9. Transport Variability in the Korea/Tsushima Strait: Characteristics and Relationship to Synoptic Atmospheric Forcing

    NASA Astrophysics Data System (ADS)

    Kang, Boonsoon; Hirose, Naoki; Fukudome, Ken-ichi

    2014-06-01

    The characteristics of high-frequency transport variation through the Korea/Tsushima Strait and its response to atmospheric synoptic forcing are investigated by using acoustic velocity measurements and a barotropic model. The first principal mode for the current data in the high-frequency band (3-50 days) is mostly coherent to transport variation through the strait. As the dominant mode, the cross-sectional pattern of the transport variability is clarified: strong axes exist in the centers of the western and eastern channels of the strait. Seasonal change in the high-frequency variability is also elucidated: it is strong from November to January, peaks in April and September, and is weak from May to August. Both data analysis and numerical experiments clarify that the transport variation is mainly attributable to wind and pressure forcing, especially in a synoptic time band (3-9 days). The amplitude of the high-frequency variability is even stronger than that of seasonal transport variation. The lagged cross-covariance reveals a spatial pattern that causes the transport variation to be a synoptic weather system passing eastward along the mid-latitudes with a period of around 5 days. The transport response to this synoptic change is investigated through numerical experiments with idealized atmospheric forcing. The experiments reveal that about 40-50% of the total transport fluctuations in the synoptic time band are explained by the idealized forcing, with a contribution from wind stress higher than that from pressure. Sensitivity experiments on the forcing area show that the pressure-driven transport fluctuation is mainly determined by forcing over the East/Japan Sea. Additional experiments indicate that the strait transport fluctuation responds coherently to the local and remote winds of the synoptic atmospheric forcing.

  10. Current transport characteristics of pSe-nMoSe2 heterojunction diode

    NASA Astrophysics Data System (ADS)

    Sumesh, C. K.; Patel, K. D.; Pathak, V. M.; Srivastava, R.

    2010-12-01

    The characteristics of heterojunction diode pSe-nMoSe2 fabricated from thermally evaporated p-Se films on n-type Molybdenum diselenide (MoSe2) grown by direct vapour transport (DVT) technique have been examined by using current-voltage measurements. To investigate the dark current transport mechanism in pSe-nMoSe2 heterojunctions the current-voltage characteristics were measured in the temperature range 100-300 K. The prepared diode shows a rectification ratio of the order of 103 within the range -2 to 2 V. A multi-step tunnelling model was used to analyze the I-V-T characteristics of the prepared device. The activation energy determined from the saturation current was about 1.16 eV.

  11. Parameter exploration of staircase-shape extracellular stimulation for targeted stimulation of myelinated axon.

    PubMed

    Ueno, Ayako; Karashima, Akihiro; Nakao, Mitsuyuki; Katayama, Norihiro

    2011-01-01

    Spatio-temporal dynamics of a mathematical model of myelinated axon in response to staircase-shape extracellular electrical stimulation, which was developed for selective nerve stimulation, is investigated by the computer simulation. It is shown that the response is classified into four types: subthreshold response, cathodic excitation, anodal block and anodal break excitation. Based on the simulation results, simple diagrams representing the response characteristics of the axon are constructed as functions of stimulation parameters and distance between the axon and electrode. The diagram would be useful for determining simulation parameters for dynamic targeted stimulation of myelinated axon.

  12. Colloid transport in dolomite rock fractures: effects of fracture characteristics, specific discharge, and ionic strength.

    PubMed

    Mondal, Pulin K; Sleep, Brent E

    2012-09-18

    The effects of fracture characteristics, specific discharge, and ionic strength on microsphere transport in variable-aperture dolomite rock fractures were studied in a laboratory-scale system. Fractures with different aperture distributions and mineral compositions were artificially created in two dolomite rock blocks. Transport tests were conducted with bromide and carboxylate-modified latex microspheres (20, 200, and 500 nm diameter). Under overall unfavorable attachment conditions, there was significant retention of the 20 nm microsphere and minimal retention of the 500 nm microsphere for all conditions examined. Aperture variability produced significant spatial variation in colloid transport. Flushing with low ionic strength solution (1 mM) following microsphere transport at 12 mM ionic strength solution produced a spike in effluent microsphere concentrations, consistent with retention of colloids in secondary energy minima. Surface roughness and charge heterogeneity effects may have also contributed to the effect of microsphere size on retention. Matrix diffusion influenced bromide transport but was not a dominant factor in transport for any microsphere size. Calibrated one-dimensional, two-site kinetic model parameters for colloid transport in fractured dolomite were sensitive to the physical and chemical properties of both the fractured dolomite and the colloids, indicating the need for mechanistic modeling for accurate prediction.

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

    PubMed Central

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

    2013-01-01

    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

  14. 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

    SciTech Connect

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

    2011-01-05

    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.

  15. Numerical modeling of current-voltage characteristics to extract transport properties of organic semiconductors

    NASA Astrophysics Data System (ADS)

    Basile, A. F.; Fraboni, B.

    2014-11-01

    The current vs. voltage (I-V) characteristics of single crystal rubrene Organic Field-Effect Transistors (OFETs) and polycrystalline poly(p-phenylenevinylene) (PPV) films are modeled using the polaron transport theory presented in a previous work [A. F. Basile et al., J. Appl. Phys. 115, 244505 (2014)]. The model is first applied to rubrene OFETs, where transport is two-dimensional and is confined near the interface between the insulator and the organic semiconductor. By considering the effect of image charges in the insulator and by assuming a constant intrinsic mobility, we reproduce both the positive and the negative temperature dependences of the channel mobilities measured on OFETs having a gate dielectric and an air-gap insulator, respectively. In addition, we adapt this model to the three-dimensional transport in PPV films, characterized by effective mobilities which depend on temperature, charge density, and electric field. We show that the I-V characteristics of these materials can be matched by the numerical solution of the Poisson and drift-diffusion equations assuming a constant intrinsic mobility. The polaron binding energy can account for the thermally activated behavior of the I-V characteristics and for the increase of the effective mobility at high applied voltages. Therefore, this model enables to extract the intrinsic transport parameters of organic semiconductors, independent of the device structure, and of the measurement conditions.

  16. Macropore system characteristics controls on non-reactive solute transport at different flow rates

    NASA Astrophysics Data System (ADS)

    Larsbo, Mats; Koestel, John

    2014-05-01

    Preferential flow and transport in macroporous soils are important pathways for the leaching of agrochemicals through soils. Preferential solute transport in soil is to a large extent determined by the macropore system characteristics and the water flow conditions. The importance of different characteristics of the macropore system is likely to vary with the flow conditions. The objective of this study was to determine which properties of the macropore system that control the shape of non-reactive tracer solute breakthrough curves at different steady-state flow rates. We sampled five undisturbed columns (20 cm high, 20 cm diameter) from the soil surface of four soils with clay contents between 21 and 50 %. Solute transport experiments were carried out under unsaturated conditions at 2, 4, 6, 8 and 12 mm h-1 flow rates. For each flow rate a pulse of potassium bromide solution was applied at the soil surface and the electrical conductivity was measured with high temporal resolution in the column effluent. We used the 5 % arrival time and the holdback factor to estimate the degree of preferential transport from the resulting breakthrough curves. Unsaturated hydraulic conductivities were measured at the soil surface of the columns using a tension disc infiltrometer. The macropore system was imaged by industrial X-ray computed tomography at a resolution of 125 μm in all directions. Measures of the macropore system characteristics including measures of pore continuity were calculated from these images using the ImageJ software. Results show that the degree of preferential transport is generally increasing with flow rate when larger pores become active in the transport. The degree of preferential flow was correlated to measures of macropore topology. This study show that conclusions drawn from experiments carried out at one flow rate should generally not be extrapolated to other flow rates.

  17. Thiazolidinediones Promote Axonal Growth through the Activation of the JNK Pathway

    PubMed Central

    Quintanilla, Rodrigo A.; Godoy, Juan A.; Alfaro, Ivan; Cabezas, Deny; von Bernhardi, Rommy; Bronfman, Miguel; Inestrosa, Nibaldo C.

    2013-01-01

    The axon is a neuronal process involved in protein transport, synaptic plasticity, and neural regeneration. It has been suggested that their structure and function are profoundly impaired in neurodegenerative diseases. Previous evidence suggest that Peroxisome Proliferator-Activated Receptors-γ (PPARγ promote neuronal differentiation on various neuronal cell types. In addition, we demonstrated that activation of PPARγby thiazolidinediones (TZDs) drugs that selectively activate PPARγ prevent neurite loss and axonal damage induced by amyloid-β (Aβ). However, the potential role of TZDs in axonal elongation and neuronal polarity has not been explored. We report here that the activation of PPARγ by TZDs promoted axon elongation in primary hippocampal neurons. Treatments with different TZDs significantly increased axonal growth and branching area, but no significant effects were observed in neurite elongation compared to untreated neurons. Treatment with PPARγ antagonist (GW 9662) prevented TZDs-induced axonal growth. Recently, it has been suggested that the c-Jun N-terminal kinase (JNK) plays an important role regulating axonal growth and neuronal polarity. Interestingly, in our studies, treatment with TZDs induced activation of the JNK pathway, and the pharmacological blockage of this pathway prevented axon elongation induced by TZDs. Altogether, these results indicate that activation of JNK induced by PPARγactivators stimulates axonal growth and accelerates neuronal polarity. These novel findings may contribute to the understanding of the effects of PPARγ on neuronal differentiation and validate the use of PPARγ activators as therapeutic agents in neurodegenerative diseases. PMID:23741474

  18. Thiazolidinediones promote axonal growth through the activation of the JNK pathway.

    PubMed

    Quintanilla, Rodrigo A; Godoy, Juan A; Alfaro, Ivan; Cabezas, Deny; von Bernhardi, Rommy; Bronfman, Miguel; Inestrosa, Nibaldo C

    2013-01-01

    The axon is a neuronal process involved in protein transport, synaptic plasticity, and neural regeneration. It has been suggested that their structure and function are profoundly impaired in neurodegenerative diseases. Previous evidence suggest that Peroxisome Proliferator-Activated Receptors-γ (PPARγ promote neuronal differentiation on various neuronal cell types. In addition, we demonstrated that activation of PPARγby thiazolidinediones (TZDs) drugs that selectively activate PPARγ prevent neurite loss and axonal damage induced by amyloid-β (Aβ). However, the potential role of TZDs in axonal elongation and neuronal polarity has not been explored. We report here that the activation of PPARγ by TZDs promoted axon elongation in primary hippocampal neurons. Treatments with different TZDs significantly increased axonal growth and branching area, but no significant effects were observed in neurite elongation compared to untreated neurons. Treatment with PPARγ antagonist (GW 9662) prevented TZDs-induced axonal growth. Recently, it has been suggested that the c-Jun N-terminal kinase (JNK) plays an important role regulating axonal growth and neuronal polarity. Interestingly, in our studies, treatment with TZDs induced activation of the JNK pathway, and the pharmacological blockage of this pathway prevented axon elongation induced by TZDs. Altogether, these results indicate that activation of JNK induced by PPARγactivators stimulates axonal growth and accelerates neuronal polarity. These novel findings may contribute to the understanding of the effects of PPARγ on neuronal differentiation and validate the use of PPARγ activators as therapeutic agents in neurodegenerative diseases.

  19. Neurofilament dot blot assays: novel means of assessing axon viability in culture.

    PubMed

    Hares, Kelly; Kemp, Kevin; Gray, Elizabeth; Scolding, Neil; Wilkins, Alastair

    2011-06-15

    Axonal structure and integrity are vital to overall neuronal maintenance and action potential propagation. Neurofilaments (NFs) are one of the main cytoskeletal components of axons and phosphorylation of NF subunits regulates speed of NF transport through axons and determines optimal axonal calibre required for signal propagation. Many previous studies of neuroprotective agents have focussed on neuronal viability in models of neurodegenerative disease, without specifically considering axon function as an indicator of neuronal damage. In this study, we have focused on developing novel assays for determining axon viability by measuring levels of neurofilament phosphorylation in cultured cortical neurons. The nitric oxide donor DETANONOate (NO) was used as an inflammatory insult and glial cell line-derived neurotrophic factor (GDNF) and superoxide dismutase (SOD) were tested as potential axonal protective agents. Using 'dot blot' methodologies, we show a decrease in NF phosphorylation in cortical neurons exposed to NO-mediated cell toxicity and an attenuation of NO-mediated changes in NF phosphorylation associated with GDNF and SOD treatment. These results correlated well with immunocytochemical counts. We propose therefore that the dot blot assay is a novel method for assessing axonal integrity in vitro and may play a useful role in the future for testing the effects of agents on axonal viability, providing a reliable and reproducible screening method for potential therapeutics for neurodegenerative diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Endogenous Nmnat2 Is an Essential Survival Factor for Maintenance of Healthy Axons

    PubMed Central

    Gilley, Jonathan; Coleman, Michael P.

    2010-01-01

    The molecular triggers for axon degeneration remain unknown. We identify endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnat1 and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured Wld S neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that WldS does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. We conclude that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and propose that, when present, the more long-lived, functionally related WldS protein substitutes for Nmnat2 loss after axon injury. Endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders. PMID:20126265

  1. Relations between macropore network characteristics and the degree of preferential solute transport

    NASA Astrophysics Data System (ADS)

    Larsbo, M.; Koestel, J.; Jarvis, N.

    2014-12-01

    The characteristics of the soil macropore network determine the potential for fast transport of agrochemicals and contaminants through the soil. The objective of this study was to examine the relationships between macropore network characteristics, hydraulic properties and state variables and measures of preferential transport. Experiments were carried out under near-saturated conditions on undisturbed columns sampled from four agricultural topsoils of contrasting texture and structure. Macropore network characteristics were computed from 3-D X-ray tomography images of the soil pore system. Non-reactive solute transport experiments were carried out at five steady-state water flow rates from 2 to 12 mm h-1. The degree of preferential transport was evaluated by the normalised 5% solute arrival time and the apparent dispersivity calculated from the resulting breakthrough curves. Near-saturated hydraulic conductivities were measured on the same samples using a tension disc infiltrometer placed on top of the columns. Results showed that many of the macropore network characteristics were inter-correlated. For example, large macroporosities were associated with larger specific macropore surface areas and better local connectivity of the macropore network. Generally, an increased flow rate resulted in earlier solute breakthrough and a shifting of the arrival of peak concentration towards smaller drained volumes. Columns with smaller macroporosities, poorer local connectivity of the macropore network and smaller near-saturated hydraulic conductivities exhibited a greater degree of preferential transport. This can be explained by the fact that, with only two exceptions, global (i.e. sample scale) continuity of the macropore network was still preserved at low macroporosities. Thus, for any given flow rate, pores of larger diameter were actively conducting solute in soils of smaller near-saturated hydraulic conductivity. This was associated with larger local transport

  2. A three-dimensional method-of-characteristics solute-transport model (MOC3D)

    USGS Publications Warehouse

    Konikow, L.F.; Goode, D.J.; Hornberger, G.Z.

    1996-01-01

    This report presents a model, MOC3D, that simulates three-dimensional solute transport in flowing ground water. The model computes changes in concentration of a single dissolved chemical constituent over time that are caused by advective transport, hydrodynamic dispersion (including both mechanical dispersion and diffusion), mixing (or dilution) from fluid sources, and mathematically simple chemical reactions (including linear sorption, which is represented by a retardation factor, and decay). The transport model is integrated with MODFLOW, a three-dimensional ground-water flow model that uses implicit finite-difference methods to solve the transient flow equation. MOC3D uses the method of characteristics to solve the transport equation on the basis of the hydraulic gradients computed with MODFLOW for a given time step. This implementation of the method of characteristics uses particle tracking to represent advective transport and explicit finite-difference methods to calculate the effects of other processes. However, the explicit procedure has several stability criteria that may limit the size of time increments for solving the transport equation; these are automatically determined by the program. For improved efficiency, the user can apply MOC3D to a subgrid of the primary MODFLOW grid that is used to solve the flow equation. However, the transport subgrid must have uniform grid spacing along rows and columns. The report includes a description of the theoretical basis of the model, a detailed description of input requirements and output options, and the results of model testing and evaluation. The model was evaluated for several problems for which exact analytical solutions are available and by benchmarking against other numerical codes for selected complex problems for which no exact solutions are available. These test results indicate that the model is very accurate for a wide range of conditions and yields minimal numerical dispersion for advection

  3. [Mechanisms of growth of neuronal axons and dendrites].

    PubMed

    Lest'anová, Z; Bacová, Z; Havránek, T; Bakos, J

    2013-01-01

    Brain development is determined by neuronal differentiation including changes of cell polarity and asymetric growth of neuronal processes. Although, there are many unkown factors contributing to changes of lenght of neuronal cones, mounting experimental and review papers focus on changes of growth conus and role of axonal transport. In particular, mechanisms of actin/microtubule polymerisation and depolymerisation are important. Role of intracellular calcium is also significant. Normal and properly timed changes of lenght of axons and dendrites are dependent on interaction of neurons and glia. Moreover, regeneration of injured axons is dependent on growth factors secreted from glial cells. The aim of the present study is characterisation of the most important mechanisms underlying changes of lenght of neurites.

  4. Axonal Localization of Integrins in the CNS Is Neuronal Type and Age Dependent

    PubMed Central

    Soleman, Sara; Mason, Matthew R. J.; Verhaagen, Joost; Bensadoun, Jean-Charles; Aebischer, Patrick

    2016-01-01

    The regenerative ability of CNS axons decreases with age, however, this ability remains largely intact in PNS axons throughout adulthood. These differences are likely to correspond with age-related silencing of proteins necessary for axon growth and elongation. In previous studies, it has been shown that reintroduction of the α9 integrin subunit (tenascin-C receptor, α9) that is downregulated in adult CNS can improve neurite outgrowth and sensory axon regeneration after a dorsal rhizotomy or a dorsal column crush spinal cord lesion. In the current study, we demonstrate that virally expressed integrins (α9, α6, or β1 integrin) in the adult rat sensorimotor cortex and adult red nucleus are excluded from axons following neuronal transduction. Attempts to stimulate transport by inclusion of a cervical spinal injury and thus an upregulation of extracellular matrix molecules at the lesion site, or cotransduction with its binding partner, β1 integrin, did not induce integrin localization within axons. In contrast, virally expressed α9 integrin in developing rat cortex (postnatal day 5 or 10) demonstrated clear localization of integrins in cortical axons revealed by the presence of integrin in the axons of the corpus callosum and internal capsule, as well as in the neuronal cell body. Furthermore, examination of dorsal root ganglia neurons and retinal ganglion cells demonstrated integrin localization both within peripheral nerve as well as dorsal root axons and within optic nerve axons, respectively. Together, our results suggest a differential ability for in vivo axonal transport of transmembrane proteins dependent on neuronal age and subtype. PMID:27570822

  5. Typology, early differentiation, and exuberant growth of a set of cortical axons.

    PubMed

    Bressoud, R; Innocenti, G M

    1999-03-29

    The corpus callosum interconnects both corresponding (homotopic) and noncorresponding (heterotopic) cortical sites of the two hemispheres. We have studied the axons that establish heterotopic connections from visual areas 17 and 18 (E axons) by using anterogradely transported biocytin and three-dimensional serial reconstructions in adult cats and in kittens. Their site of termination distinguished four types of axons. Type EI ends near the border between areas 19/21a or 7, and type EII near the PMLS/PLLS border (posteromedial and posterolateral lateral suprasylvian areas). Type EIII and EIV terminate the first near the PMLS/PLLS and PMLS/21a borders, and the second near the PMLS/PLLS and 19/21a or 7 borders. Taking into account the previously studied homotopic axons (O axons; Houzel et al. [1994] Eur. J. Neurosci. 6:898-917), it can be concluded that areas 17 and 18 are interhemispherically connected by at least five types of axons, three of which (O, EI, and EII) terminate near one areal border, the other two (types EIII and EIV), near two areal borders. All types terminate near representations of the vertical meridian of the visual field. The different types of axons can be identified already during the first postnatal week; at this age, unlike in the adult, they originate not only near the 17/18 border, but also, transiently, in area 17. This suggests that the developing cortex contains sets of neurons destined to send their axon to different targets; however, the axons grow beyond their sites of adult termination. Indeed, exuberant growth takes place at the stage of axonal elongation, and at subsequent stages of axonal differentiation, i.e., during subcortical branching, intracortical branching and synaptogenesis. The growth is progressively more constrained in its topographic distribution and the axons are subsequently reshaped by regressive events.

  6. Development of Systematic Approaches for Calibration of Subsurface Transport Models Using Hard and Soft Data on System Characteristics and Behavior

    DTIC Science & Technology

    2011-02-02

    environmental problems to include the simulation of contaminant transport and volatilization in the shallow subsurface and water content fluctuation...REPORT Development of systematic approaches for calibration of subsurface transport models using hard and soft data on system characteristics and...Park, NC 27709-2211 15. SUBJECT TERMS model calibration, data assimilation, subsurface transport Tissa H. Illangasekare, Toshihiro Sakaki Colorado

  7. ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis.

    PubMed

    Konopacki, Filip A; Wong, Hovy Ho-Wai; Dwivedy, Asha; Bellon, Anaïs; Blower, Michael D; Holt, Christine E

    2016-04-01

    Endocytosis and local protein synthesis (LPS) act coordinately to mediate the chemotropic responses of axons, but the link between these two processes is poorly understood. The endosomal sorting complex required for transport (ESCRT) is a key regulator of cargo sorting in the endocytic pathway, and here we have investigated the role of ESCRT-II, a critical ESCRT component, in Xenopus retinal ganglion cell (RGC) axons. We show that ESCRT-II is present in RGC axonal growth cones (GCs) where it co-localizes with endocytic vesicle GTPases and, unexpectedly, with the Netrin-1 receptor, deleted in colorectal cancer (DCC). ESCRT-II knockdown (KD) decreases endocytosis and, strikingly, reduces DCC in GCs and leads to axon growth and guidance defects. ESCRT-II-depleted axons fail to turn in response to a Netrin-1 gradient in vitro and many axons fail to exit the eye in vivo These defects, similar to Netrin-1/DCC loss-of-function phenotypes, can be rescued in whole (in vitro) or in part (in vivo) by expressing DCC. In addition, ESCRT-II KD impairs LPS in GCs and live imaging reveals that ESCRT-II transports mRNAs in axons. Collectively, our results show that the ESCRT-II-mediated endocytic pathway regulates both DCC and LPS in the axonal compartment and suggest that ESCRT-II aids gradient sensing in GCs by coupling endocytosis to LPS.

  8. Transcellular degradation of axonal mitochondria

    PubMed Central

    Davis, Chung-ha O.; Kim, Keun-Young; Bushong, Eric A.; Mills, Elizabeth A.; Boassa, Daniela; Shih, Tiffany; Kinebuchi, Mira; Phan, Sebastien; Zhou, Yi; Bihlmeyer, Nathan A.; Nguyen, Judy V.; Jin, Yunju; Ellisman, Mark H.; Marsh-Armstrong, Nicholas

    2014-01-01

    It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons. Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered. PMID:24979790

  9. Mechanisms of Distal Axonal Degeneration in Peripheral Neuropathies

    PubMed Central

    Cashman, Christopher R.; Höke, Ahmet

    2015-01-01

    Peripheral neuropathy is a common complication of a variety of diseases and treatments, including diabetes, cancer chemotherapy, and infectious causes (HIV, hepatitis C, and Campylobacter jejuni). Despite the fundamental difference between these insults, peripheral neuropathy develops as a combination of just six primary mechanisms: altered metabolism, covalent modification, altered organelle function and reactive oxygen species formation, altered intracellular and inflammatory signaling, slowed axonal transport, and altered ion channel dynamics and expression. All of these pathways converge to lead to axon dysfunction and symptoms of neuropathy. The detailed mechanisms of axon degeneration itself have begun to be elucidated with studies of animal models with altered degeneration kinetics, including the slowed Wallerian degeneration (Wlds) and Sarmknockout animal models. These studies have shown axonal degeneration to occur througha programmed pathway of injury signaling and cytoskeletal degradation. Insights into the common disease insults that converge on the axonal degeneration pathway promise to facilitate the development of therapeutics that may be effective against other mechanisms of neurodegeneration. PMID:25617478

  10. Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism.

    PubMed

    Saab, Aiman S; Tzvetavona, Iva D; Trevisiol, Andrea; Baltan, Selva; Dibaj, Payam; Kusch, Kathrin; Möbius, Wiebke; Goetze, Bianka; Jahn, Hannah M; Huang, Wenhui; Steffens, Heinz; Schomburg, Eike D; Pérez-Samartín, Alberto; Pérez-Cerdá, Fernando; Bakhtiari, Davood; Matute, Carlos; Löwel, Siegrid; Griesinger, Christian; Hirrlinger, Johannes; Kirchhoff, Frank; Nave, Klaus-Armin

    2016-07-06

    Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Morphological and molecular features of the mammalian olfactory sensory neuron axons: What makes these axons so special?

    PubMed

    Nedelec, Stéphane; Dubacq, Caroline; Trembleau, Alain

    2005-03-01

    The main organization and gross morphology of the mammalian olfactory primary pathway, from the olfactory epithelium to the olfactory bulb, has been initially characterized using classical anatomical and ultrastructural approaches. During the last fifteen years, essentially thanks to the cloning of the odorant receptor genes, and to the characterization of a number of molecules expressed by the olfactory sensory neuron axons and their environment, significant new insights have been gained into the understanding of the development and adult functioning of this system. In the course of these genetic, biochemical and neuroanatomical studies, however, several molecular and structural features were uncovered that appear somehow to be unique to these axons. For example, these axons express odorant receptors in their terminal segment, and transport several mRNA species and at least two transcription factors. In the present paper, we review these unusual structural and molecular features and speculate about their possible functions in the development and maintenance of the olfactory system.

  12. Large-voltage behavior of charge transport characteristics in nanosystems with weak electron–vibration coupling

    PubMed Central

    Belzig, Wolfgang

    2015-01-01

    Summary We study analytically the Full Counting Statistics of the charge transport through a nanosystem consisting of a few electronic levels weakly coupled to a discrete vibrational mode. In the limit of large transport voltage bias the cumulant generating function can be evaluated explicitly based solely on the intuitive physical arguments and classical master equation description of the vibration mode. We find that for the undamped vibrational modes mutual dynamical interplay between electronic and vibronic degrees of freedom leads to strongly nonlinear (in voltage) transport characteristics of the nanosystem. In particular, we find that for large voltages the k-th cumulant of the current grows as V 2k to be contrasted with the linear dependence in case of more strongly externally damped and thus thermalized vibrational modes. PMID:26425436

  13. Electrical transport properties and current density - voltage characteristic of PVA-Ag nanocomposite film

    NASA Astrophysics Data System (ADS)

    Das, A. K.; Dutta, B.; Sinha, S.; Mukherjee, A.; Basu, S.; Meikap, A. K.

    2016-05-01

    Silver (Ag) nanoparticle and Polyvinyl alcohol (PVA) - Silver (Ag) composite have been prepared and its dielectric constant, ac conductivity, and current density-voltage characteristics have been studied, at and above room temperature. Here correlated barrier hopping found to be the dominant charge transport mechanism with maximum barrier height of 0.11 eV. The sample, under ±5 V applied voltage, show back to back Schottky diode behaviour.

  14. Significance of transcytosis in Alzheimer's disease: BACE1 takes the scenic route to axons

    PubMed Central

    Buggia-Prévot, Virginie; Thinakaran, Gopal

    2015-01-01

    Neurons have developed elaborate mechanisms for sorting of proteins to their destination in dendrites and axons as well as dynamic local trafficking. Recent evidence suggests that polarized axonal sorting of β-site converting enzyme 1 (BACE1), a type I transmembrane aspartyl protease involved in Alzheimer's disease (AD) pathogenesis, entails an unusual journey. In hippocampal neurons, BACE1 internalized from dendrites is conveyed in recycling endosomes via unidirectional retrograde transport towards the soma and sorted to axons where BACE1 becomes enriched. In comparison to other transmembrane proteins that undergo transcytosis or elimination in somatodendritic compartment, vectorial transport of internalized BACE1 in dendrites is unique and intriguing. Dysfunction of protein transport contributes to pathogenesis of AD and other neurodegenerative diseases. Therefore, characterization of BACE1 transcytosis is an important addition to the multiple lines of evidence that highlight the crucial role played by endosomal trafficking pathway as well as axonal sorting mechanisms in AD pathogenesis. PMID:26126792

  15. Slow axoplasmic transport under scrutiny.

    PubMed

    Court, Felipe A; Alvarez, Jaime

    2011-01-01

    The origin of axoplasmic proteins is central for the biology of axons. For over fifty years axons have been considered unable to synthesize proteins and that cell bodies supply them with proteins by a slow transport mechanism. To allow for prolonged transport times, proteins were assumed to be stable, i.e., not degraded in axons. These are now textbook notions that configure the slow transport model (STM). The aim of this article is to cast doubts on the validity of STM, as a step toward gaining more understanding about the supply of axoplasmic proteins. First, the stability of axonal proteins claimed by STM has been disproved by experimental evidence. Moreover, the evidence for protein synthesis in axons indicates that the repertoire is extensive and the amount sizeable, which disproves the notion that axons are unable to synthesize proteins and that cell bodies supply most axonal proteins. In turn, axoplasmic protein synthesis gives rise to the metabolic model (MM). We point out a few inconsistencies in STM that MM redresses. Although both models address the supply of proteins to axons, so far they have had no crosstalk. Since proteins underlie every conceivable cellular function, it is necessary to re-evaluate in-depth the origin of axonal proteins. We hope this will shape a novel understanding of the biology of axons, with impact on development and maintenance of axons, nerve repair, axonopathies and plasticity, to mention a few fields.

  16. AQUAPORIN-1 WATER PERMEABILITY AS A NOVEL DETERMINANT OF AXONAL REGENERATION IN DORSAL ROOT GANGLION NEURONS

    PubMed Central

    Zhang, Hua; Verkman, A.S.

    2015-01-01

    Dorsal root ganglion (DRG) neurons transduce peripheral pain signals through small-diameter, non-myelinated C-fibers, which, when injured, can regenerate to restore pain sensation. Water channel aquaporin-1 (AQP1) is expressed at the plasma membrane of cell bodies and axons of DRG neurons, where it modulates the sensing of certain types of pain. Here, we found that AQP1 is also involved in DRG axonal growth and regeneration by a mechanism that may involve water transport-facilitated extension of axonal outgrowths. Spontaneous and nerve growth factor-stimulated axonal extension was reduced in cultures of AQP1-deficient DRG neurons and DRG explants compared to the wildtype. Axonal growth in AQP1-deficient DRG cultures was rescued by transfection with AQP1 or a different water-transporting AQP (AQP4), but not by a non-water-transporting AQP1 mutant. Following sciatic nerve compression injury AQP1 expression was increased in DRG neurons in wildtype mice, and DRG axonal growth was impaired in AQP1-deficient mice. Our results indicate AQP1 as a novel determinant of DRG axonal regeneration and hence a potential therapeutic target to accelerate neuronal regeneration. PMID:25585012

  17. Aquaporin-1 water permeability as a novel determinant of axonal regeneration in dorsal root ganglion neurons.

    PubMed

    Zhang, Hua; Verkman, A S

    2015-03-01

    Dorsal root ganglion (DRG) neurons transduce peripheral pain signals through small-diameter, non-myelinated C-fibers, which, when injured, can regenerate to restore pain sensation. Water channel aquaporin-1 (AQP1) is expressed at the plasma membrane of cell bodies and axons of DRG neurons, where it modulates the sensing of certain types of pain. Here, we found that AQP1 is also involved in DRG axonal growth and regeneration by a mechanism that may involve water transport-facilitated extension of axonal outgrowths. Spontaneous and nerve growth factor-stimulated axonal extension was reduced in cultures of AQP1-deficient DRG neurons and DRG explants compared to the wildtype. Axonal growth in AQP1-deficient DRG cultures was rescued by transfection with AQP1 or a different water-transporting AQP (AQP4), but not by a non-water-transporting AQP1 mutant. Following sciatic nerve compression injury AQP1 expression was increased in DRG neurons in wildtype mice, and DRG axonal growth was impaired in AQP1-deficient mice. Our results indicate AQP1 as a novel determinant of DRG axonal regeneration and hence a potential therapeutic target to accelerate neuronal regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Effect of cell physicochemical characteristics and motility on bacterial transport in groundwater

    USGS Publications Warehouse

    Becker, M.W.; Collins, S.A.; Metge, D.W.; Harvey, R.W.; Shapiro, A.M.

    2004-01-01

    The influence of physicochemical characteristics and motility on bacterial transport in groundwater were examined in flow-through columns. Four strains of bacteria isolated from a crystalline rock groundwater system were investigated, with carboxylate-modified and amidine-modified latex microspheres and bromide as reference tracers. The bacterial isolates included a gram-positive rod (ML1), a gram-negative motile rod (ML2), a nonmotile mutant of ML2 (ML2m), and a gram-positive coccoid (ML3). Experiments were repeated at two flow velocities, in a glass column packed with glass beads, and in another packed with iron-oxyhydroxide coated glass beads. Bacteria breakthrough curves were interpreted using a transport equation that incorporates a sorption model from microscopic observation of bacterial deposition in flow-cell experiments. The model predicts that bacterial desorption rate will decrease exponentially with the amount of time the cell is attached to the solid surface. Desorption kinetics appeared to influence transport at the lower flow rate, but were not discernable at the higher flow rate. Iron-oxyhydroxide coatings had a lower-than-expected effect on bacterial breakthrough and no effect on the microsphere recovery in the column experiments. Cell wall type and shape also had minor effects on breakthrough. Motility tended to increase the adsorption rate, and decrease the desorption rate. The transport model predicts that at field scale, desorption rate kinetics may be important to the prediction of bacteria transport rates. ?? 2003 Elsevier B.V. All rights reserved.

  19. Effect of cell physicochemical characteristics and motility on bacterial transport in groundwater

    NASA Astrophysics Data System (ADS)

    Becker, Matthew W.; Collins, Samantha A.; Metge, David W.; Harvey, Ronald W.; Shapiro, Allen M.

    2004-04-01

    The influence of physicochemical characteristics and motility on bacterial transport in groundwater were examined in flow-through columns. Four strains of bacteria isolated from a crystalline rock groundwater system were investigated, with carboxylate-modified and amidine-modified latex microspheres and bromide as reference tracers. The bacterial isolates included a gram-positive rod (ML1), a gram-negative motile rod (ML2), a nonmotile mutant of ML2 (ML2m), and a gram-positive coccoid (ML3). Experiments were repeated at two flow velocities, in a glass column packed with glass beads, and in another packed with iron-oxyhydroxide coated glass beads. Bacteria breakthrough curves were interpreted using a transport equation that incorporates a sorption model from microscopic observation of bacterial deposition in flow-cell experiments. The model predicts that bacterial desorption rate will decrease exponentially with the amount of time the cell is attached to the solid surface. Desorption kinetics appeared to influence transport at the lower flow rate, but were not discernable at the higher flow rate. Iron-oxyhydroxide coatings had a lower-than-expected effect on bacterial breakthrough and no effect on the microsphere recovery in the column experiments. Cell wall type and shape also had minor effects on breakthrough. Motility tended to increase the adsorption rate, and decrease the desorption rate. The transport model predicts that at field scale, desorption rate kinetics may be important to the prediction of bacteria transport rates.

  20. Effect of cell physicochemical characteristics and motility on bacterial transport in groundwater.

    PubMed

    Becker, Matthew W; Collins, Samantha A; Metge, David W; Harvey, Ronald W; Shapiro, Allen M

    2004-04-01

    The influence of physicochemical characteristics and motility on bacterial transport in groundwater were examined in flow-through columns. Four strains of bacteria isolated from a crystalline rock groundwater system were investigated, with carboxylate-modified and amidine-modified latex microspheres and bromide as reference tracers. The bacterial isolates included a gram-positive rod (ML1), a gram-negative motile rod (ML2), a nonmotile mutant of ML2 (ML2m), and a gram-positive coccoid (ML3). Experiments were repeated at two flow velocities, in a glass column packed with glass beads, and in another packed with iron-oxyhydroxide coated glass beads. Bacteria breakthrough curves were interpreted using a transport equation that incorporates a sorption model from microscopic observation of bacterial deposition in flow-cell experiments. The model predicts that bacterial desorption rate will decrease exponentially with the amount of time the cell is attached to the solid surface. Desorption kinetics appeared to influence transport at the lower flow rate, but were not discernable at the higher flow rate. Iron-oxyhydroxide coatings had a lower-than-expected effect on bacterial breakthrough and no effect on the microsphere recovery in the column experiments. Cell wall type and shape also had minor effects on breakthrough. Motility tended to increase the adsorption rate, and decrease the desorption rate. The transport model predicts that at field scale, desorption rate kinetics may be important to the prediction of bacteria transport rates.

  1. Transport characteristics and origins of carbon monoxide and ozone in Hong Kong, South China

    NASA Astrophysics Data System (ADS)

    Ding, Aijun; Wang, Tao; Fu, Congbin

    2013-08-01

    characteristics and origins of carbon monoxide (CO) and ozone (O3) transported to Hong Kong were investigated using backward Lagrangian particle dispersion modeling (LPDM) and measurement data collected at a regional background station, Hok Tsui, in South China during 2005-2007. LPDM was conducted using the Hybrid Single-Particle Lagrangian Integrated Trajectory model (HYSPLIT) driven by Global Data Assimilation System (GDAS) data and emission inventories of CO. The LPDM simulations showed very good performance in characterizing temporal variations in CO on both seasonal and synoptic scales during the 3 years. Based on the LPDM simulations, the seasonal cycles of the source origins and transport age of CO were investigated. Carbon monoxide at the background station was found to originate mainly from the coastal regions in southern China and from eastern China, with averaged transport time of about 1 day and 2-4 days, respectively. The O3 annual maximum in autumn was influenced by regional transport of O3 precursors originating from eastern China, suggesting a strong continental outflow and its regional impacts. Photochemical processes contributed to occurrence of O3 episodes with sharp daytime O3 peaks produced from fresh subregional emissions from Hong Kong and the Pearl River Delta region, mixed with inland continental air. By examining the O3-CO relationship and Lagrangian transport time, the roles of transport and photochemical processes in different seasons at Hok Tsui were also investigated. This study demonstrated unique applications of backward LPDM in observational data analysis as well as in understanding source-receptor relationships and multiscale transport of CO and O3.

  2. Characteristics and Outcomes of Patients Injured in Road Traffic Crashes and Transported by Emergency Medical Services.

    PubMed

    Huang, Chun-Ying; Rau, Cheng-Shyuan; Chuang, Jung-Fang; Kuo, Pao-Jen; Hsu, Shiun-Yuan; Chen, Yi-Chun; Hsieh, Hsiao-Yun; Hsieh, Ching-Hua

    2016-02-19

    To investigate the injury characteristics and mortality of patients transported by emergency medical services (EMS) and hospitalized for trauma following a road traffic crash, data obtained from the Trauma Registry System were retrospectively reviewed for trauma admissions between 1 January 2009 and 31 December 2013 in a Level I trauma center. Of 16,548 registered patients, 3978 and 1440 patients injured in road traffic crashes were transported to the emergency department by EMS and non-EMS, respectively. Patients transported by EMS had lower Glasgow coma scale (GCS) scores and worse hemodynamic measures. Compared to patients transported by non-EMS, more patients transported by EMS required procedures (intubation, chest tube insertion, and blood transfusion) at the emergency department. They also sustained a higher injury severity, as measured by the injury severity score (ISS) and the new injury severity score (NISS). Lastly, in-hospital mortality was higher among the EMS than the non-EMS group (1.8% vs. 0.3%, respectively; p < 0.001). However, we found no statistically significant difference in the adjusted odds ratio (AOR) for mortality among patients transported by EMS after adjustment for ISS (AOR 4.9, 95% CI 0.33-2.26), indicating that the higher incidence of mortality was likely attributed to the patients' higher injury severity. In addition, after propensity score matching, logistic regression of 58 well-matched pairs did not show a significant influence of transportation by EMS on mortality (OR: 0.578, 95% CI: 0.132-2.541 p = 0.468).

  3. Characteristics and Outcomes of Patients Injured in Road Traffic Crashes and Transported by Emergency Medical Services

    PubMed Central

    Huang, Chun-Ying; Rau, Cheng-Shyuan; Chuang, Jung-Fang; Kuo, Pao-Jen; Hsu, Shiun-Yuan; Chen, Yi-Chun; Hsieh, Hsiao-Yun; Hsieh, Ching-Hua

    2016-01-01

    To investigate the injury characteristics and mortality of patients transported by emergency medical services (EMS) and hospitalized for trauma following a road traffic crash, data obtained from the Trauma Registry System were retrospectively reviewed for trauma admissions between 1 January 2009 and 31 December 2013 in a Level I trauma center. Of 16,548 registered patients, 3978 and 1440 patients injured in road traffic crashes were transported to the emergency department by EMS and non-EMS, respectively. Patients transported by EMS had lower Glasgow coma scale (GCS) scores and worse hemodynamic measures. Compared to patients transported by non-EMS, more patients transported by EMS required procedures (intubation, chest tube insertion, and blood transfusion) at the emergency department. They also sustained a higher injury severity, as measured by the injury severity score (ISS) and the new injury severity score (NISS). Lastly, in-hospital mortality was higher among the EMS than the non-EMS group (1.8% vs. 0.3%, respectively; p < 0.001). However, we found no statistically significant difference in the adjusted odds ratio (AOR) for mortality among patients transported by EMS after adjustment for ISS (AOR 4.9, 95% CI 0.33–2.26), indicating that the higher incidence of mortality was likely attributed to the patients’ higher injury severity. In addition, after propensity score matching, logistic regression of 58 well-matched pairs did not show a significant influence of transportation by EMS on mortality (OR: 0.578, 95% CI: 0.132–2.541 p = 0.468). PMID:26907318

  4. Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons