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Sample records for axonal mitochondrial transport

  1. 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

  2. Dynamics of Mitochondrial Transport in Axons.

    PubMed

    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

  3. Demyelination increases axonal stationary mitochondrial size and the speed of axonal mitochondrial transport

    PubMed Central

    Kiryu-Seo, Sumiko; Ohno, Nobuhiko; Kidd, Grahame J.; Komuro, Hitoshi; Trapp, Bruce D.

    2010-01-01

    Axonal degeneration contributes to permanent neurological disability in inherited and acquired diseases of myelin. Mitochondrial dysfunction has been proposed as a major contributor to this axonal degeneration. It remains to be determined, however, if myelination, demyelination or remyelination alter the size and distribution of axonal mitochondrial stationary sites or the rates of axonal mitochondrial transport. Using live myelinated rat dorsal root ganglion (DRG) cultures, we investigated whether myelination and lysolecithin-induced demyelination affect axonal mitochondria. Myelination increased the size of axonal stationary mitochondrial sites by 2.3 fold. Following demyelination, the size of axonal stationary mitochondrial sites was increased by an additional 2.2 fold and the transport velocity of motile mitochondria was increased by 47%. These measures returned to the levels of myelinated axons following remyelination. Demyelination induced activating transcription factor (ATF) 3 in DRG neurons. Knockdown of neuronal ATF3 by shRNA abolished the demyelination-induced increase in axonal mitochondrial transport and increased nitrotyrosine immunoreactivity in axonal mitochondria, suggesting that neuronal ATF3 expression and increased mitochondrial transport protect demyelinated axons from oxidative damage. In response to insufficient ATP production, demyelinated axons increase the size of stationary mitochondrial sites and thereby balance ATP production with the increased energy needs of nerve conduction. PMID:20463228

  4. 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

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

    PubMed

    Zhou, Bing; Yu, Panpan; Lin, Mei-Yao; Sun, Tao; Chen, Yanmin; Sheng, Zu-Hang

    2016-07-01

    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

  6. Imaging Axonal Transport of Mitochondria

    PubMed Central

    Wang, Xinnan; Schwarz, Thomas L.

    2010-01-01

    Neuronal mitochondria need to be transported and distributed in axons and dendrites in order to ensure an adequate energy supply and provide sufficient Ca2+ buffering in each portion of these highly extended cells. Errors in mitochondrial transport are implicated in neurodegenerative diseases. Here we present useful tools to analyze axonal transport of mitochondria both in vitro in cultured rat neurons and in vivo in Drosophila larval neurons. These methods enable investigators to take advantage of both systems to study the properties of mitochondrial motility under normal or pathological conditions. PMID:19426876

  7. 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

  8. Mitochondrial biogenesis is required for axonal growth.

    PubMed

    Vaarmann, Annika; Mandel, Merle; Zeb, Akbar; Wareski, Przemyslaw; Liiv, Joanna; Kuum, Malle; Antsov, Eva; Liiv, Mailis; Cagalinec, Michal; Choubey, Vinay; Kaasik, Allen

    2016-06-01

    During early development, neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. Rapid growth requires a large quantity of building materials, efficient intracellular transport and also a considerable amount of energy. To produce this energy, the neuron should first generate new mitochondria because the pre-existing mitochondria are unlikely to provide a sufficient acceleration in ATP production. Here, we demonstrate that mitochondrial biogenesis and ATP production are required for axonal growth and neuronal development in cultured rat cortical neurons. We also demonstrate that growth signals activating the CaMKKβ, LKB1-STRAD or TAK1 pathways also co-activate the AMPK-PGC-1α-NRF1 axis leading to the generation of new mitochondria to ensure energy for upcoming growth. In conclusion, our results suggest that neurons are capable of signalling for upcoming energy requirements. Earlier activation of mitochondrial biogenesis through these pathways will accelerate the generation of new mitochondria, thereby ensuring energy-producing capability for when other factors for axonal growth are synthesized. PMID:27122166

  9. Mitochondrial immobilization mediated by syntaphilin facilitates survival of demyelinated axons

    PubMed Central

    Ohno, Nobuhiko; Chiang, Hao; Mahad, Don J.; Kidd, Grahame J.; Liu, LiPing; Ransohoff, Richard M.; Sheng, Zu-Hang; Komuro, Hitoshi; Trapp, Bruce D.

    2014-01-01

    Axonal degeneration is a primary cause of permanent neurological disability in individuals with the CNS demyelinating disease multiple sclerosis. Dysfunction of axonal mitochondria and imbalanced energy demand and supply are implicated in degeneration of chronically demyelinated axons. The purpose of this study was to define the roles of mitochondrial volume and distribution in axonal degeneration following acute CNS demyelination. We show that the axonal mitochondrial volume increase following acute demyelination of WT CNS axons does not occur in demyelinated axons deficient in syntaphilin, an axonal molecule that immobilizes stationary mitochondria to microtubules. These findings were supported by time-lapse imaging of WT and syntaphilin-deficient axons in vitro. When demyelinated, axons deficient in syntaphilin degenerate at a significantly greater rate than WT axons, and this degeneration can be rescued by reducing axonal electrical activity with the Na+ channel blocker flecainide. These results support the concept that syntaphilin-mediated immobilization of mitochondria to microtubules is required for the volume increase of axonal mitochondria following acute demyelination and protects against axonal degeneration in the CNS. PMID:24958879

  10. Calcium Release from Intra-Axonal Endoplasmic Reticulum Leads to Axon Degeneration through Mitochondrial Dysfunction

    PubMed Central

    Villegas, Rosario; Martinez, Nicolas W.; Lillo, Jorge; Pihan, Phillipe; Hernandez, Diego; Twiss, Jeffery L.

    2014-01-01

    Axonal degeneration represents an early pathological event in neurodegeneration, constituting an important target for neuroprotection. Regardless of the initial injury, which could be toxic, mechanical, metabolic, or genetic, degeneration of axons shares a common mechanism involving mitochondrial dysfunction and production of reactive oxygen species. Critical steps in this degenerative process are still unknown. Here we show that calcium release from the axonal endoplasmic reticulum (ER) through ryanodine and IP3 channels activates the mitochondrial permeability transition pore and contributes to axonal degeneration triggered by both mechanical and toxic insults in ex vivo and in vitro mouse and rat model systems. These data reveal a critical and early ER-dependent step during axonal degeneration, providing novel targets for axonal protection in neurodegenerative conditions. PMID:24849352

  11. NDE1 and GSK3β Associate with TRAK1 and Regulate Axonal Mitochondrial Motility: Identification of Cyclic AMP as a Novel Modulator of Axonal Mitochondrial Trafficking.

    PubMed

    Ogawa, Fumiaki; Murphy, Laura C; Malavasi, Elise L V; O'Sullivan, Shane T; Torrance, Helen S; Porteous, David J; Millar, J Kirsty

    2016-05-18

    Mitochondria are essential for neuronal function, providing the energy required to power neurotransmission, and fulfilling many important additional roles. In neurons, mitochondria must be efficiently transported to sites, including synapses, where their functions are required. Neurons, with their highly elongated morphology, are consequently extremely sensitive to defective mitochondrial trafficking which can lead to neuronal ill-health/death. We recently demonstrated that DISC1 associates with mitochondrial trafficking complexes where it associates with the core kinesin and dynein adaptor molecule TRAK1. We now show that the DISC1 interactors NDE1 and GSK3β also associate robustly with TRAK1 and demonstrate that NDE1 promotes retrograde axonal mitochondrial movement. GSK3β is known to modulate axonal mitochondrial motility, although reports of its actual effect are conflicting. We show that, in our system, GSK3β promotes anterograde mitochondrial transport. Finally, we investigated the influence of cAMP elevation upon mitochondrial motility, and found a striking increase in mitochondrial motility and retrograde movement. DISC1, NDE1, and GSK3β are implicated as risk factors for major mental illness. Our demonstration that they function together within mitochondrial trafficking complexes suggests that defective mitochondrial transport may be a contributory disease mechanism in some cases of psychiatric disorder. PMID:26815013

  12. Characterization of mitochondrial transport in neurons.

    PubMed

    Zhou, Bing; Lin, Mei-Yao; Sun, Tao; Knight, Adam L; Sheng, Zu-Hang

    2014-01-01

    Mitochondria are cellular power plants that supply ATP to power various biological activities essential for neuronal growth, survival, and function. Due to extremely varied morphological features, neurons face exceptional challenges to maintain energy homeostasis. Neurons require specialized mechanisms distributing mitochondria to distal synapses where energy is in high demand. Axons and synapses undergo activity-dependent remodeling, thereby altering mitochondrial distribution. The uniform microtubule polarity has made axons particularly useful for exploring mechanisms regulating mitochondrial transport. Mitochondria alter their motility under stress conditions or when their integrity is impaired. Therefore, research into the mechanisms regulating mitochondrial motility in healthy and diseased neurons is an important emerging frontier in neurobiology. In this chapter, we discuss the current protocols in the characterization of axonal mitochondrial transport in primary neuron cultures isolated from embryonic rats and adult mice. We also briefly discuss new procedures developed in our lab in analyzing mitochondrial motility patterns at presynaptic terminals and evaluate their impact on synaptic vesicle release. PMID:25416353

  13. Regulation of Axonal Transport by Protein Kinases.

    PubMed

    Gibbs, Katherine L; Greensmith, Linda; Schiavo, Giampietro

    2015-10-01

    The intracellular transport of organelles, proteins, lipids, and RNA along the axon is essential for neuronal function and survival. This process, called axonal transport, is mediated by two classes of ATP-dependent motors, kinesins, and cytoplasmic dynein, which carry their cargoes along microtubule tracks. Protein kinases regulate axonal transport through direct phosphorylation of motors, adapter proteins, and cargoes, and indirectly through modification of the microtubule network. The misregulation of axonal transport by protein kinases has been implicated in the pathogenesis of several nervous system disorders. Here, we review the role of protein kinases acting directly on axonal transport and discuss how their deregulation affects neuronal function, paving the way for the exploitation of these enzymes as novel drug targets. PMID:26410600

  14. A model for fast axonal transport.

    PubMed

    Blum, J J; Reed, M C

    1985-01-01

    A model for fast axonal transport is developed in which the essential features are that organelles may interact with mechanochemical cross-bridges that in turn interact with microtubules, forming an organelle-engine-microtubule complex which is transported along the microtubules. Computer analysis of the equations derived to describe such a system show that most of the experimental observations on fast axonal transport can be simulated by the model, indicating that the model is useful for the interpretation and design of experiments aimed at clarifying the mechanism of fast axonal transport. PMID:2416456

  15. 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

  16. Dysregulation of the Axonal Trafficking of Nuclear-encoded Mitochondrial mRNA alters Neuronal Mitochondrial Activity and Mouse Behavior

    PubMed Central

    Kar, Amar N.; Sun, Ching-Yu; Reichard, Kathryn; Gervasi, Noreen M.; Pickel, James; Nakazawa, Kazu; Gioio, Anthony E.; Kaplan, Barry B.

    2014-01-01

    Local translation of nuclear-encoded mitochondrial mRNAs is essential for mitochondrial activity, yet there is little insight into the role that axonal trafficking of these transcripts play in neuronal function and behavior. Previously, we identified a 38 nucleotide stem-loop structure (zipcode) in the 3′ untranslated region of the Cytochrome C oxidase IV (COXIV) mRNA that directs the transport of a reporter mRNA to the axon of superior cervical ganglion neurons (SCG). Over-expression of a chimeric reporter mRNA with the COXIV zipcode competed with the axonal trafficking of endogenous COXIV mRNA, and led to attenuated axon growth in SCG neurons. Here, we show that exogenous expression of the COXIV zipcode in cultured SCG neurons also results in the reduction of local ATP levels and increases levels of reactive oxygen species (ROS) in the axon. We took advantage of this “competition” phenotype to investigate the in vivo significance of axonal transport of COXIV mRNA. Towards this end, we generated transgenic mice expressing a fluorescent reporter fused to COXIV zipcode under a forebrain-specific promoter. Immunohistological analyses and RT-PCR analyses of RNA from the transgenic mouse brain showed expression of the reporter in the deep layer neurons in the pre-frontal and frontal cortex. Consistent with the in vitro studies, we observed increased ROS levels in neurons of these transgenic animals. A battery of behavioral tests on transgenic mice expressing the COXIV zipcode revealed an “anxiety-like” behavioral phenotype, suggesting an important role for axonal trafficking of nuclear-encoded mitochondrial mRNAs in neuronal physiology and animal behavior. PMID:24151253

  17. 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

  18. Axonal transport of ribonucleoprotein particles (vaults).

    PubMed

    Li, J Y; Volknandt, W; Dahlstrom, A; Herrmann, C; Blasi, J; Das, B; Zimmermann, H

    1999-01-01

    RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon

  19. Regulation of Mitochondrial Transport in Neurons

    PubMed Central

    Lin, Mei-Yao; Sheng, Zu-Hang

    2015-01-01

    Mitochondria are cellular power plants that supply ATP to power various biological activities essential for neuronal growth, survival, and function. Due to unique morphological features, neurons face exceptional challenges to maintain ATP and Ca2+ homeostasis. Neurons require specialized mechanisms distributing mitochondria to distal areas where energy and Ca2+ buffering are in high demand, such as synapses and axonal branches. These distal compartments also undergo development- and activity-dependent remodeling, thereby altering mitochondrial trafficking and distribution. Mitochondria move bi-directionally, pause briefly, and move again, frequently changing direction. In mature neurons, only one-third of axonal mitochondria are motile. Stationary mitochondria serve as local energy sources and buffer intracellular Ca2+. The balance between motile and stationary mitochondria responds quickly to changes in axonal and synaptic physiology. Furthermore, neurons are postmitotic cells surviving for the lifetime of the organism; thus, mitochondria need to be removed when they become aged or dysfunction. Mitochondria also alter their motility under stress conditions or when their integrity is impaired. Therefore, regulation of mitochondrial transport is essential to meet altered metabolic requirements and to remove aged and damaged mitochondria or replenish healthy ones to distal terminals. Defects in mitochondrial transport and altered distribution are implicated in the pathogenesis of several major neurological disorders. Thus, research into the mechanisms regulating mitochondrial motility is an important emerging frontier in neurobiology. This short review provides an updated overview on motor-adaptor machineries that drive and regulate mitochondrial transport and docking receptors that anchor axonal mitochondria in response to the changes in synaptic activity, metabolic requirement, and altered mitochondrial integrity. The review focuses on microtubule (MT

  20. A model of axonal transport drug delivery

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Andrey

    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.

  1. 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".

  2. 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

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

    PubMed

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

    2015-02-01

    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

  4. Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults

    PubMed Central

    Lassus, Benjamin; Magifico, Sebastien; Pignon, Sandra; Belenguer, Pascale; Miquel, Marie-Christine; Peyrin, Jean-Michel

    2016-01-01

    In chronic neurodegenerative syndromes, neurons progressively die through a generalized retraction pattern triggering retrograde axonal degeneration toward the cell bodies, which molecular mechanisms remain elusive. Recent observations suggest that direct activation of pro-apoptotic signaling in axons triggers local degenerative events associated with early alteration of axonal mitochondrial dynamics. This raises the question of the role of mitochondrial dynamics on both axonal vulnerability stress and their implication in the spreading of damages toward unchallenged parts of the neuron. Here, using microfluidic chambers, we assessed the consequences of interfering with OPA1 and DRP1 proteins on axonal degeneration induced by local application of rotenone. We found that pharmacological inhibition of mitochondrial fission prevented axonal damage induced by rotenone, in low glucose conditions. While alteration of mitochondrial dynamics per se did not lead to spontaneous axonal degeneration, it dramatically enhanced axonal vulnerability to rotenone, which had no effect in normal glucose conditions, and promoted retrograde spreading of axonal degeneration toward the cell body. Altogether, our results suggest a mitochondrial priming effect in axons as a key process of axonal degeneration. In the context of neurodegenerative diseases, like Parkinson’s and Alzheimer’s, mitochondria fragmentation could hasten neuronal death and initiate spatial dispersion of locally induced degenerative events. PMID:27604820

  5. Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults.

    PubMed

    Lassus, Benjamin; Magifico, Sebastien; Pignon, Sandra; Belenguer, Pascale; Miquel, Marie-Christine; Peyrin, Jean-Michel

    2016-01-01

    In chronic neurodegenerative syndromes, neurons progressively die through a generalized retraction pattern triggering retrograde axonal degeneration toward the cell bodies, which molecular mechanisms remain elusive. Recent observations suggest that direct activation of pro-apoptotic signaling in axons triggers local degenerative events associated with early alteration of axonal mitochondrial dynamics. This raises the question of the role of mitochondrial dynamics on both axonal vulnerability stress and their implication in the spreading of damages toward unchallenged parts of the neuron. Here, using microfluidic chambers, we assessed the consequences of interfering with OPA1 and DRP1 proteins on axonal degeneration induced by local application of rotenone. We found that pharmacological inhibition of mitochondrial fission prevented axonal damage induced by rotenone, in low glucose conditions. While alteration of mitochondrial dynamics per se did not lead to spontaneous axonal degeneration, it dramatically enhanced axonal vulnerability to rotenone, which had no effect in normal glucose conditions, and promoted retrograde spreading of axonal degeneration toward the cell body. Altogether, our results suggest a mitochondrial priming effect in axons as a key process of axonal degeneration. In the context of neurodegenerative diseases, like Parkinson's and Alzheimer's, mitochondria fragmentation could hasten neuronal death and initiate spatial dispersion of locally induced degenerative events. PMID:27604820

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

    PubMed

    Godzik, Katharina; Coleman, Michael P

    2015-04-01

    The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases. PMID:25352062

  7. Differential extraction of axonally transported proteoglycans

    SciTech Connect

    Elam, J.S. )

    1990-10-01

    Axonally transported proteoglycans were differentially solubilized by a sequence of extractions designed to infer their relationship to nerve terminal membranes. Groups of goldfish were injected unilaterally with 35SO4 and contralateral optic tecta containing axonally transported molecules were removed 16 h later. Tecta were homogenized in isotonic buffer and centrifuged at 100,000 g for 60 min to create a total supernatant fraction. Subsequent homogenizations followed by recentrifugation were with hypotonic buffer (lysis extract), 1 M NaCl, Triton X-100 or alternatively Triton-1 M NaCl. Populations of proteoglycans in each extract were isolated on DEAE ion exchange columns and evaluated for content of glycosaminoglycans (GAGs). Results show the distribution of transported proteoglycans to be 26.3% total soluble, 13.7% lysis extract, 13.8% NaCl extract, 12.2% Triton extract, and 46.2% Triton-NaCl extract. Proteoglycans from all fractions contained heparan sulfate as the predominant GAG, with lesser amounts of chondroitin (4 or 6) sulfate. The possible localizations of transported proteoglycans suggested by the extraction results are discussed.

  8. Axon diameter and axonal transport: In vivo and in vitro effects of androgens

    PubMed Central

    Pesaresi, M; Soon-Shiong, R; French, L; Kaplan, DR; Miller, FD; Paus, T.

    2015-01-01

    Testosterone is a sex hormone involved in brain maturation via multiple molecular mechanisms. Previous human studies described age-related changes in the overall volume and morphological properties of white matter during male puberty. Based on this work, we have proposed that testosterone may induce an increase of radial growth and, possibly, modulate axonal transport. In order to determine whether this is the case we have used two different experimental approaches. With electron microscopy, we have evaluated sex differences in the structural properties of axons in the corpus callosum (splenium) of young rats, and tested consequences of castration carried out after weaning. Then we examined in vitro the effect of the non-aromatizable androgen Mibolerone on the structure and bidirectional transport of wheat-germ agglutinin vesicles in the axons of cultured sympathetic neurons. With electron microscopy, we found robust sex differences in axonal diameter (males>females) and g ratio (males>females). Removal of endogenous testosterone by castration was associated with lower axon diameter and lower g ratio in castrated (vs. intact) males. In vitro, Mibolerone influenced the axonal transport in a time- and dose-dependent manner, and increased the axon caliber as compared with vehicle-treated neurons. These findings are consistent with the role of testosterone in shaping the axon by regulating its radial growth, as predicted by the initial human studies. PMID:25956809

  9. Quantitative Analysis of Axonal Transport by Using Compartmentalized and Surface Micropatterned Culture of Neurons

    PubMed Central

    2012-01-01

    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. PMID:24358503

  10. Kinetic properties of normal and perturbed axonal transport of serotonin in a single identified axon.

    PubMed Central

    Goldberg, D J; Schwartz, J H; Sherbany, A A

    1978-01-01

    1. The axonal transport of pulses of [3H]serotonin was studied in an axon of the serotonergic giant cerebral neurone (GCN) of Aplysia californica. 2. [3H]serotonin was transported as a discrete peak which was followed by a relatively low, smooth trail. 3. The peak broadened as it moved along the axon, sometimes skewing in the proximal direction. 4. The velocity of the transport was highly dependent on temperature, but the rate of peak broadening was not. The velocity was 130 mm per day at 23 degrees C and 48 mm per day at 14 degrees C. The rate of broadening was 143 micrometer per mm transport at 23 degrees C and 156 micrometer per mm transport at 14 degrees C. 5. In another series of experiments, almost the entire length of the lip nerve, which contained the axon of GCN, was maintained at 1--3 degrees C to block transport. The GCN's cell body and the proximal few millimetres of the nerve were maintained at 23 degrees C. As a result, the amount of [3H]serotonin in the proximal segment of the nerve increased manyfold during periods of up to 4 hr. The concentrated pulse of [3H]serotonin resulting from this treatment was transported more slowly than normal after the cooling was terminated. Sometimes, a minor peak split from the major peak of radioactivity and was transported a normal velocity. 6. Incubation of the cerebral ganglion and nerves for 16 hr in the presence of anisomycin, an inhibitor of protein synthesis, reduced by nearly fourfold the amount of [3H]serotonin subsequently exported into the axon of the GCN. The transport velocity at this reduced concentration was less than half the normal value. If the concentration of [3H]serotonin in the axon was restored to normal in the presence of anisomycin, the velocity of transport was also returned to normal. 7. We conclude that the velocity of transport of serotonergic vesicles in the axon of the GCN is positively dependent on the local concentration of vesicles, except at very high concentrations, where the

  11. Schwann cell mitochondrial metabolism supports long-term axonal survival and peripheral nerve function

    PubMed Central

    Viader, Andreu; Golden, Judith P.; Baloh, Robert H.; Schmidt, Robert E.; Hunter, Daniel A.; Milbrandt, Jeffrey

    2011-01-01

    Mitochondrial dysfunction is a common cause of peripheral neuropathies. While the role of neuron and axonal mitochondria in peripheral nerve disease is well appreciated, whether Schwann cell (SC) mitochondrial deficits contribute to peripheral neuropathies is unclear. Here we examine how SC mitochondrial dysfunction affects axonal survival and contributes to the decline of peripheral nerve function by generating mice with SC-specific mitochondrial deficits. These mice (Tfam-SCKOs) were produced through the tissue-specific deletion of the mitochondrial transcription factor A gene (Tfam), which is essential for mitochondrial DNA (mtDNA) transcription and maintenance. Tfam-SCKOs were viable but, as they aged, they developed a progressive peripheral neuropathy characterized by nerve conduction abnormalities as well as extensive muscle denervation. Morphological examination of Tfam-SCKO nerves revealed early preferential loss of small unmyelinated fibers followed by prominent demyelination and degeneration of larger-caliber axons. Tfam-SCKOs displayed sensory and motor deficits consistent with this pathology. Remarkably, the severe mtDNA depletion and respiratory chain abnormalities in Tfam-SCKO mice did not affect SC proliferation or survival. Mitochondrial function in SCs is therefore essential for maintenance of axonal survival and normal peripheral nerve function, suggesting that SC mitochondrial dysfunction contributes to human peripheral neuropathies. PMID:21752989

  12. Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.

    PubMed Central

    Elluru, R G; Bloom, G S; Brady, S T

    1995-01-01

    The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport. Images PMID:7538359

  13. 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).

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

    PubMed Central

    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. PMID:26790099

  15. Reducing Lissencephaly-1 levels augments mitochondrial transport and has a protective effect in adult Drosophila neurons

    PubMed Central

    Vagnoni, Alessio; Hoffmann, Patrick C.; Bullock, Simon L.

    2016-01-01

    ABSTRACT Defective transport of mitochondria in axons is implicated in the pathogenesis of several age-associated neurodegenerative diseases. However, the regulation and function of axonal mitochondrial motility during normal ageing is poorly understood. Here, we use novel imaging procedures to characterise axonal transport of these organelles in the adult Drosophila wing nerve. During early adult life there is a boost and progressive decline in the proportion of mitochondria that are motile, which is not due to general changes in cargo transport. Experimental inhibition of the mitochondrial transport machinery specifically in adulthood accelerates the appearance of focal protein accumulations in ageing axons, which is suggestive of defects in protein homeostasis. Unexpectedly, lowering levels of Lissencephaly-1 (Lis1), a dynein motor co-factor, augments axonal mitochondrial transport in ageing wing neurons. Lis1 mutations suppress focal protein accumulations in ageing neurons, including those caused by interfering with the mitochondrial transport machinery. Our data provide new insights into the dynamics of mitochondrial motility in adult neurons in vivo, identify Lis1 as a negative regulator of transport of these organelles, and provide evidence of a link between mitochondrial movement and neuronal protein homeostasis. PMID:26598558

  16. Mitochondrial Dynamics Decrease Prior to Axon Degeneration Induced by Vincristine and are Partially Rescued by Overexpressed cytNmnat1

    PubMed Central

    Berbusse, Gregory W.; Woods, Laken C.; Vohra, Bhupinder P. S.; Naylor, Kari

    2016-01-01

    Axon degeneration is a prominent feature of various neurodegenerative diseases, such as Parkinson’s and Alzheimer’s, and is often characterized by aberrant mitochondrial dynamics. Mitochondrial fission, fusion, and motility have been shown to be particularly important in progressive neurodegeneration. Thus we investigated these imperative dynamics, as well as mitochondrial fragmentation in vincristine induced axon degradation in cultured dorsal root ganglia (DRG) neurons. CytNmnat1 inhibits axon degeneration in various paradigms including vincristine toxicity. The mechanism of its protection is not yet fully understood; therefore, we also investigated the effect of cytNmnat1 on mitochondrial dynamics in vincristine treated neurons. We observed that vincristine treatment decreases the rate of mitochondrial fission, fusion and motility and induces mitochondrial fragmentation. These mitochondrial events precede visible axon degeneration. Overexpression of cytNmnat1 inhibits axon degeneration and preserves the normal mitochondrial dynamics and motility in vincristine treated neurons. We suggest the alterations in mitochondrial structure and dynamics are early events which lead to axon degeneration and cytNmnat1 blocks axon degeneration by halting the vincristine induced changes to mitochondrial structure and dynamics. PMID:27486387

  17. Mitochondrial Dynamics Decrease Prior to Axon Degeneration Induced by Vincristine and are Partially Rescued by Overexpressed cytNmnat1.

    PubMed

    Berbusse, Gregory W; Woods, Laken C; Vohra, Bhupinder P S; Naylor, Kari

    2016-01-01

    Axon degeneration is a prominent feature of various neurodegenerative diseases, such as Parkinson's and Alzheimer's, and is often characterized by aberrant mitochondrial dynamics. Mitochondrial fission, fusion, and motility have been shown to be particularly important in progressive neurodegeneration. Thus we investigated these imperative dynamics, as well as mitochondrial fragmentation in vincristine induced axon degradation in cultured dorsal root ganglia (DRG) neurons. CytNmnat1 inhibits axon degeneration in various paradigms including vincristine toxicity. The mechanism of its protection is not yet fully understood; therefore, we also investigated the effect of cytNmnat1 on mitochondrial dynamics in vincristine treated neurons. We observed that vincristine treatment decreases the rate of mitochondrial fission, fusion and motility and induces mitochondrial fragmentation. These mitochondrial events precede visible axon degeneration. Overexpression of cytNmnat1 inhibits axon degeneration and preserves the normal mitochondrial dynamics and motility in vincristine treated neurons. We suggest the alterations in mitochondrial structure and dynamics are early events which lead to axon degeneration and cytNmnat1 blocks axon degeneration by halting the vincristine induced changes to mitochondrial structure and dynamics. PMID:27486387

  18. 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

  19. A dynamical system model of neurofilament transport in axons

    PubMed Central

    Craciun, Gheorghe; Brown, Anthony; Friedman, Avner

    2007-01-01

    We develop a dynamical system model for the transport of neurofilaments in axons, inspired by Brown’s “stop-and-go” model for slow axonal transport. We use fast/slow time-scale arguments to lower the number of relevant parameters in our model. Then, we use experimental data of Wang and Brown to estimate all but one parameter. We show that we can choose this last remaining parameter such that the results of our model agree with pulse-labeling experiments from three different nerve cell types, and also agree with stochastic simulation results. PMID:15975597

  20. Axon Transport and Neuropathy: Relevant Perspectives on the Etiopathogenesis of Familial Dysautonomia.

    PubMed

    Tourtellotte, Warren G

    2016-03-01

    Peripheral neuropathies are highly prevalent and are most often associated with chronic disease, side effects from chemotherapy, or toxic-metabolic abnormalities. Neuropathies are less commonly caused by genetic mutations, but studies of the normal function of mutated proteins have identified particular vulnerabilities that often implicate mitochondrial dynamics and axon transport mechanisms. Hereditary sensory and autonomic neuropathies are a group of phenotypically related diseases caused by monogenic mutations that primarily affect sympathetic and sensory neurons. Here, I review evidence to indicate that many genetic neuropathies are caused by abnormalities in axon transport. Moreover, in hereditary sensory and autonomic neuropathies. There may be specific convergence on gene mutations that disrupt nerve growth factor signaling, upon which sympathetic and sensory neurons critically depend. PMID:26724390

  1. The myriad roles of Miro in the nervous system: axonal transport of mitochondria and beyond

    PubMed Central

    Lee, Kyu-Sun; Lu, Bingwei

    2014-01-01

    Mitochondrial rho GTPase (Miro) is a mitochondrial outer membrane protein containing two GTPase domains and two helix-loop-helix Ca2+-binding domains called EF hands. Pioneering genetic studies in Drosophila first revealed a key function of Miro in regulating the axonal transport of mitochondria, during which Miro forms a multi-protein transport complex with Milton and Kinesin heavy chain (KHC) to link trafficking mitochondria with the microtubule (MT) cytoskeleton. Recent studies showed that through binding to the EF hands of Miro and causing conformational changes of Miro and alteration of protein-protein interactions within the transport complex, Ca2+ can alter the engagement of mitochondria with the MT/kinesin network, offering one mechanism to match mitochondrial distribution with neuronal activity. Despite the importance of the Miro/Milton/Kinesin complex in regulating mitochondrial transport in metazoans, not all components of the transport complex are conserved in lower organisms, and transport-independent functions of Miro are emerging. Here we review the diverse functions of the evolutionarily conserved Miro proteins that are relevant to the development, maintenance, and functioning of the nervous system and discuss the potential contribution of Miro dysfunction to the pathogenesis of diseases of the nervous system. PMID:25389385

  2. Elevated Mitochondrial Bioenergetics and Axonal Arborization Size Are Key Contributors to the Vulnerability of Dopamine Neurons.

    PubMed

    Pacelli, Consiglia; Giguère, Nicolas; Bourque, Marie-Josée; Lévesque, Martin; Slack, Ruth S; Trudeau, Louis-Éric

    2015-09-21

    Although the mechanisms underlying the loss of neurons in Parkinson's disease are not well understood, impaired mitochondrial function and pathological protein aggregation are suspected as playing a major role. Why DA (dopamine) neurons and a select small subset of brain nuclei are particularly vulnerable to such ubiquitous cellular dysfunctions is presently one of the key unanswered questions in Parkinson's disease research. One intriguing hypothesis is that their heightened vulnerability is a consequence of their elevated bioenergetic requirements. Here, we show for the first time that vulnerable nigral DA neurons differ from less vulnerable DA neurons such as those of the VTA (ventral tegmental area) by having a higher basal rate of mitochondrial OXPHOS (oxidative phosphorylation), a smaller reserve capacity, a higher density of axonal mitochondria, an elevated level of basal oxidative stress, and a considerably more complex axonal arborization. Furthermore, we demonstrate that reducing axonal arborization by acting on axon guidance pathways with Semaphorin 7A reduces in parallel the basal rate of mitochondrial OXPHOS and the vulnerability of nigral DA neurons to the neurotoxic agents MPP(+) (1-methyl-4-phenylpyridinium) and rotenone. Blocking L-type calcium channels with isradipine was protective against MPP(+) but not rotenone. Our data provide the most direct demonstration to date in favor of the hypothesis that the heightened vulnerability of nigral DA neurons in Parkinson's disease is directly due to their particular bioenergetic and morphological characteristics. PMID:26320949

  3. 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.

  4. Disrupted-in-schizophrenia 1 (DISC1) and Syntaphilin collaborate to modulate axonal mitochondrial anchoring.

    PubMed

    Park, Cana; Lee, Seol-Ae; Hong, Ji-Ho; Suh, Yeongjun; Park, Sung Jin; Suh, Bo Kyoung; Woo, Youngsik; Choi, Jinhyuk; Huh, Ji-Won; Kim, You-Me; Park, Sang Ki

    2016-01-01

    In neuronal axons, the ratio of motile-to-stationary mitochondria is tightly regulated by neuronal activation, thereby meeting the need for local calcium buffering and maintaining the ATP supply. However, the molecular players and detailed regulatory mechanisms behind neuronal mitochondrial movement are not completely understood. Here, we found that neuronal activation-induced mitochondrial anchoring is regulated by Disrupted-in-schizophrenia 1 (DISC1), which is accomplished by functional association with Syntaphilin (SNPH). DISC1 deficiency resulted in reduced axonal mitochondrial movement, which was partially reversed by concomitant SNPH depletion. In addition, a SNPH deletion mutant lacking the sequence for interaction with DISC1 exhibited an enhanced mitochondrial anchoring effect than wild-type SNPH. Moreover, upon neuronal activation, mitochondrial movement was preserved by DISC1 overexpression, not showing immobilized response of mitochondria. Taken together, we propose that DISC1 in association with SNPH is a component of a modulatory complex that determines mitochondrial anchoring in response to neuronal activation. PMID:27370822

  5. Mitochondrial redox and pH signaling occurs in axonal and synaptic organelle clusters

    PubMed Central

    Breckwoldt, Michael O.; Armoundas, Antonis A.; Aon, Miguel A.; Bendszus, Martin; O’Rourke, Brian; Schwarzländer, Markus; Dick, Tobias P.; Kurz, Felix T.

    2016-01-01

    Redox switches are important mediators in neoplastic, cardiovascular and neurological disorders. We recently identified spontaneous redox signals in neurons at the single mitochondrion level where transients of glutathione oxidation go along with shortening and re-elongation of the organelle. We now have developed advanced image and signal-processing methods to re-assess and extend previously obtained data. Here we analyze redox and pH signals of entire mitochondrial populations. In total, we quantified the effects of 628 redox and pH events in 1797 mitochondria from intercostal axons and neuromuscular synapses using optical sensors (mito-Grx1-roGFP2; mito-SypHer). We show that neuronal mitochondria can undergo multiple redox cycles exhibiting markedly different signal characteristics compared to single redox events. Redox and pH events occur more often in mitochondrial clusters (medium cluster size: 34.1 ± 4.8 μm2). Local clusters possess higher mitochondrial densities than the rest of the axon, suggesting morphological and functional inter-mitochondrial coupling. We find that cluster formation is redox sensitive and can be blocked by the antioxidant MitoQ. In a nerve crush paradigm, mitochondrial clusters form sequentially adjacent to the lesion site and oxidation spreads between mitochondria. Our methodology combines optical bioenergetics and advanced signal processing and allows quantitative assessment of entire mitochondrial populations. PMID:27000952

  6. PATHOLOGIES OF AXONAL TRANSPORT IN NEURODEGENERATIVE DISEASES

    PubMed Central

    Liu, Xin-An; Rizzo, Valerio; Puthanveettil, Sathyanarayanan V.

    2013-01-01

    Gene products such as organelles, proteins and RNAs are actively transported to synaptic terminals for the remodeling of pre-existing neuronal connections and formation of new ones. Proteins described as molecular motors mediate this transport and utilize specialized cytoskeletal proteins that function as molecular tracks for the motor based transport of cargos. Molecular motors such as kinesins and dynein's move along microtubule tracks formed by tubulins whereas myosin motors utilize tracks formed by actin. Deficits in active transport of gene products have been implicated in a number of neurological disorders. We describe such disorders collectively as “transportopathies”. Here we review current knowledge of critical components of active transport and their relevance to neurodegenerative diseases. PMID:23750323

  7. Impairment of SOD1-G93A motility is linked to mitochondrial movement in axons of hippocampal neurons.

    PubMed

    Bae, Jae Ryul; Kim, Sung Hyun

    2016-08-01

    Superoxide dismutase 1 (SOD1) is a well-known antioxidant enzyme. Mutation of SOD1 is closely associated with the pathogenesis of neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer's disease. However, the pathologic pathways linking neurodegenerative diseases with mutation of SOD1 remain elusive. Here, we investigated the motility of SOD1-WT and -G93A (a pathogenic mutant of SOD1), and observed correlation of axonal transport of the mutant protein with mitochondria in primary cultured hippocampal neurons. The SOD1-G93A mutant showed significant accumulation at vGlut1-positive synaptic boutons and in cell bodies, compared to SOD1-WT. The proportions of motile WT and G93A proteins were similar (~30 %) while the motility velocity of SOD1-G93A was significantly slower (~40 %) than that of the WT counterpart. This motility defect of SOD1-G93A was highly correlated with mitochondrial movement. Our results collectively suggest that the SOD1-G93A mutant has a defect in motility that is linked to mitochondrial transport in axons. PMID:27464601

  8. In vivo imaging of axonal transport in murine motor and sensory neurons

    PubMed Central

    Gibbs, Katherine L.; Kalmar, Bernadett; Sleigh, James N.; Greensmith, Linda; Schiavo, Giampietro

    2016-01-01

    Background Axonal transport is essential for neuronal function and survival. Defects in axonal transport have been identified as an early pathological feature in several disorders of the nervous system. The visualisation and quantitative analysis of axonal transport in vivo in rodent models of neurological disease is therefore crucial to improve our understanding of disease pathogenesis and for the identification of novel therapeutics. New method Here, we describe a method for the in vivo imaging of axonal transport of signalling endosomes in the sciatic nerve of live, anaesthetised mice. Results This method allows the multiparametric, quantitative analysis of in vivo axonal transport in motor and sensory neurons of adult mice in control conditions and during disease progression. Comparison with existing methods Previous in vivo imaging of the axonal transport of signalling endosomes has been limited to studies in nerve explant preparations or non-invasive approaches using magnetic resonance imaging; techniques that are hampered by major drawbacks such as tissue damage and low temporal and spatial resolution. This new method allows live imaging of the axonal transport of single endosomes in the sciatic nerve in situ and a more sensitive analysis of axonal transport kinetics than previous approaches. Conclusions The method described in this paper allows an in-depth analysis of the characteristics of axonal transport in both motor and sensory neurons in vivo. It enables the detailed study of alterations in axonal transport in rodent models of neurological diseases and can be used to identify novel pharmacological modifiers of axonal transport. PMID:26424507

  9. Live imaging of mitochondrial dynamics in CNS dopaminergic neurons in vivo demonstrates early reversal of mitochondrial transport following MPP(+) exposure.

    PubMed

    Dukes, April A; Bai, Qing; Van Laar, Victor S; Zhou, Yangzhong; Ilin, Vladimir; David, Christopher N; Agim, Zeynep S; Bonkowsky, Joshua L; Cannon, Jason R; Watkins, Simon C; Croix, Claudette M St; Burton, Edward A; Berman, Sarah B

    2016-11-01

    Extensive convergent evidence collectively suggests that mitochondrial dysfunction is central to the pathogenesis of Parkinson's disease (PD). Recently, changes in the dynamic properties of mitochondria have been increasingly implicated as a key proximate mechanism underlying neurodegeneration. However, studies have been limited by the lack of a model in which mitochondria can be imaged directly and dynamically in dopaminergic neurons of the intact vertebrate CNS. We generated transgenic zebrafish in which mitochondria of dopaminergic neurons are labeled with a fluorescent reporter, and optimized methods allowing direct intravital imaging of CNS dopaminergic axons and measurement of mitochondrial transport in vivo. The proportion of mitochondria undergoing axonal transport in dopaminergic neurons decreased overall during development between 2days post-fertilization (dpf) and 5dpf, at which point the major period of growth and synaptogenesis of the relevant axonal projections is complete. Exposure to 0.5-1.0mM MPP(+) between 4 and 5dpf did not compromise zebrafish viability or cause detectable changes in the number or morphology of dopaminergic neurons, motor function or monoaminergic neurochemistry. However, 0.5mM MPP(+) caused a 300% increase in retrograde mitochondrial transport and a 30% decrease in anterograde transport. In contrast, exposure to higher concentrations of MPP(+) caused an overall reduction in mitochondrial transport. This is the first time mitochondrial transport has been observed directly in CNS dopaminergic neurons of a living vertebrate and quantified in a PD model in vivo. Our findings are compatible with a model in which damage at presynaptic dopaminergic terminals causes an early compensatory increase in retrograde transport of compromised mitochondria for degradation in the cell body. These data are important because manipulation of early pathogenic mechanisms might be a valid therapeutic approach to PD. The novel transgenic lines and

  10. 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

  11. MIRO GTPases in Mitochondrial Transport, Homeostasis and Pathology

    PubMed Central

    Tang, Bor Luen

    2015-01-01

    The evolutionarily-conserved mitochondrial Rho (MIRO) small GTPase is a Ras superfamily member with three unique features. It has two GTPase domains instead of the one found in other small GTPases, and it also has two EF hand calcium binding domains, which allow Ca2+-dependent modulation of its activity and functions. Importantly, it is specifically associated with the mitochondria and via a hydrophobic transmembrane domain, rather than a lipid-based anchor more commonly found in other small GTPases. At the mitochondria, MIRO regulates mitochondrial homeostasis and turnover. In metazoans, MIRO regulates mitochondrial transport and organization at cellular extensions, such as axons, and, in some cases, intercellular transport of the organelle through tunneling nanotubes. Recent findings have revealed a myriad of molecules that are associated with MIRO, particularly the kinesin adaptor Milton/TRAK, mitofusin, PINK1 and Parkin, as well as the endoplasmic reticulum-mitochondria encounter structure (ERMES) complex. The mechanistic aspects of the roles of MIRO and its interactors in mitochondrial homeostasis and transport are gradually being revealed. On the other hand, MIRO is also increasingly associated with neurodegenerative diseases that have roots in mitochondrial dysfunction. In this review, I discuss what is currently known about the cellular physiology and pathophysiology of MIRO functions. PMID:26729171

  12. Automated kymograph analysis for profiling axonal transport of secretory granules.

    PubMed

    Mukherjee, Amit; Jenkins, Brian; Fang, Cheng; Radke, Richard J; Banker, Gary; Roysam, Badrinath

    2011-06-01

    This paper describes an automated method to profile the velocity patterns of small organelles (BDNF granules) being transported along a selected section of axon of a cultured neuron imaged by time-lapse fluorescence microscopy. Instead of directly detecting the granules as in conventional tracking, the proposed method starts by generating a two-dimensional spatio-temporal map (kymograph) of the granule traffic along an axon segment. Temporal sharpening during the kymograph creation helps to highlight granule movements while suppressing clutter due to stationary granules. A voting algorithm defined over orientation distribution functions is used to refine the locations and velocities of the granules. The refined kymograph is analyzed using an algorithm inspired from the minimum set cover framework to generate multiple motion trajectories of granule transport paths. The proposed method is computationally efficient, robust to significant levels of noise and clutter, and can be used to capture and quantify trends in transport patterns quickly and accurately. When evaluated on a collection of image sequences, the proposed method was found to detect granule movement events with 94% recall rate and 82% precision compared to a time-consuming manual analysis. Further, we present a study to evaluate the efficacy of velocity profiling by analyzing the impact of oxidative stress on granule transport in which the fully automated analysis correctly reproduced the biological conclusion generated by manual analysis. PMID:21330183

  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. PMID:16719791

  14. Mitochondrial alarmins released by degenerating motor axon terminals activate perisynaptic Schwann cells

    PubMed Central

    Duregotti, Elisa; Negro, Samuele; Scorzeto, Michele; Zornetta, Irene; Dickinson, Bryan C.; Chang, Christopher J.; Montecucco, Cesare; Rigoni, Michela

    2015-01-01

    An acute and highly reproducible motor axon terminal degeneration followed by complete regeneration is induced by some animal presynaptic neurotoxins, representing an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that nerve terminals exposed to spider or snake presynaptic neurotoxins degenerate as a result of calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons release signaling molecules derived from mitochondria: hydrogen peroxide, mitochondrial DNA, and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells cocultured with neurons and at neuromuscular junction in vivo through the MAPK pathway. We propose that this inter- and intracellular signaling is involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases. PMID:25605902

  15. 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.

  16. Abnormal intermediate filament organization alters mitochondrial motility in giant axonal neuropathy fibroblasts

    PubMed Central

    Lowery, Jason; Jain, Nikhil; Kuczmarski, Edward R.; Mahammad, Saleemulla; Goldman, Anne; Gelfand, Vladimir I.; Opal, Puneet; Goldman, Robert D.

    2016-01-01

    Giant axonal neuropathy (GAN) is a rare disease caused by mutations in the GAN gene, which encodes gigaxonin, an E3 ligase adapter that targets intermediate filament (IF) proteins for degradation in numerous cell types, including neurons and fibroblasts. The cellular hallmark of GAN pathology is the formation of large aggregates and bundles of IFs. In this study, we show that both the distribution and motility of mitochondria are altered in GAN fibroblasts and this is attributable to their association with vimentin IF aggregates and bundles. Transient expression of wild-type gigaxonin in GAN fibroblasts reduces the number of IF aggregates and bundles, restoring mitochondrial motility. Conversely, silencing the expression of gigaxonin in control fibroblasts leads to changes in IF organization similar to that of GAN patient fibroblasts and a coincident loss of mitochondrial motility. The inhibition of mitochondrial motility in GAN fibroblasts is not due to a global inhibition of organelle translocation, as lysosome motility is normal. Our findings demonstrate that it is the pathological changes in IF organization that cause the loss of mitochondrial motility. PMID:26700320

  17. 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

  18. The kinematics of turnaround and retrograde axonal transport.

    PubMed

    Snyder, R E

    1986-11-01

    Rapid axonal transport of a pulse of 35S-methionine-labelled material was studied in vitro in the sensory neurons of amphibian sciatic nerve using a position-sensitive detector. For 10 nerves studied at 23.0 +/- 0.2 degrees C it was found that a pulse moved in the anterograde direction characterized by front edge, peak, and trailing edge transport rates of (mm/d) 180.8 +/- 2.2 (+/- SEM), 176.6 +/- 2.3, and 153.7 +/- 3.0, respectively. Following its arrival at a distal ligature, a smaller pulse was observed to move in the retrograde direction characterized by front edge and peak transport rates of 158.0 +/- 7.3 and 110.3 +/- 3.5, respectively, indicating that retrograde transport proceeds at a rate of 0.88 +/- 0.04 that of anterograde. The retrograde pulse was observed to disperse at a rate greater than the anterograde. Reversal of radiolabel at the distal ligature began 1.49 +/- 0.15 h following arrival of the first radiolabel. Considerable variation was seen between preparations in the way radiolabel accumulated in the end (ligature) regions of the nerve. Although a retrograde pulse was seen in all preparations, in 7 of 10 preparations there was no evidence of this pulse accumulating within less than 2-3 mm of a proximal ligature; however, accumulation was observed within less than 5 mm in all preparations. PMID:2432169

  19. Axonal transport of proteoglycans to the goldfish optic tectum

    SciTech Connect

    Ripellino, J.A.; Elam, J.S.

    1988-05-01

    The study addressed the question of whether /sup 35/SO/sub 4/ labeled molecules that have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a soluble fraction (soluble after centrifugation at 105,000 g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000 g) and a final 105,000 g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatography was resolved into a fraction of sulfated glycoproteins eluting at 0-0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32-0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated /sup 35/SO/sub 4/, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the results support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.

  20. Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly.

    PubMed Central

    Penfold, M E; Armati, P; Cunningham, A L

    1994-01-01

    To examine the transmission of herpes simplex virus (HSV) from axon to epidermal cell, an in vitro model was constructed consisting of human fetal dorsal root ganglia cultured in the central chamber of a dual-chamber tissue culture system separated from autologous skin explants in an exterior chamber by concentric steel cylinders adhering to the substratum through silicon grease and agarose. Axons grew through the agarose viral diffusion barrier and terminated on epidermal cells in the exterior chamber. After inoculation of HSV onto dorsal root ganglia, anterograde axonal transport of glycoprotein and nucleocapsid antigen was observed by confocal microscopy to appear in exterior chamber axons within 12 h and in epidermal cells within 16 h, moving at 2-3 mm/h. Although both enveloped and unenveloped nucleocapsids were observed in the neuronal soma by transmission electron microscopy, only nucleocapsids were observed in the axons, closely associated with microtubules. Nodule formation at the surface of HSV-infected axons, becoming more dense at the axon terminus on epidermal cells, and patches of axolemmal HSV glycoprotein D expression were observed by scanning (immuno)electron microscopy, probably representing virus emerging from the axolemma. These findings strongly suggest a specialized mode of viral transport, assembly, and egress in sensory neurons: microtubule-associated intermediate-fast anterograde axonal transport of unenveloped nucleocapsids with separate transport of glycoproteins to the distal regions of the axon and assembly prior to virus emergence at the axon terminus. Images PMID:7517552

  1. HUMMR, a hypoxia- and HIF-1α–inducible protein, alters mitochondrial distribution and transport

    PubMed Central

    Li, Yan; Lim, Seung; Hoffman, David; Aspenstrom, Pontus; Federoff, Howard J.

    2009-01-01

    Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 α (HIF-1α). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states. PMID:19528298

  2. β-Tubulin mutations that cause severe neuropathies disrupt axonal transport

    PubMed Central

    Niwa, Shinsuke; Takahashi, Hironori; Hirokawa, Nobutaka

    2013-01-01

    Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β-tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β-tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β-tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations. PMID:23503589

  3. β-Tubulin mutations that cause severe neuropathies disrupt axonal transport.

    PubMed

    Niwa, Shinsuke; Takahashi, Hironori; Hirokawa, Nobutaka

    2013-05-15

    Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β-tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β-tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β-tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations. PMID:23503589

  4. DIRECT MEASUREMENT OF FAST AXONAL ORGANELLE TRANSPORT IN THE SCIATIC NERVE OF RATS TREATED WITH ACRYLAMIDE

    EPA Science Inventory

    The effects of acrylamide on fast axonal transport have been measured primarily using the indirect methods of isotope or enzyme accumulation. e report the first direct evaluation of the effects of sub-chronic acrylamide dosing (150, 300 or 500 mg/kg total dose) on the fast axonal...

  5. 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. PMID:11741313

  6. RETROGRADE AXONAL TRANSPORT OF PHOSPHOINOSITIDES AFTER INTRANEURAL INJECTION OF [3H]MYO-INOSITOL INTO THE RAT SCIATIC NERVE

    EPA Science Inventory

    Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection (Gould, 1976; Gould et at., 1987b), retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precurso...

  7. Pulse exposure of cultured rat neurons to aluminum-maltol affected the axonal transport system.

    PubMed

    Kashiwagi, Y; Nakamura, Y; Miyamae, Y; Hashimoto, R; Takeda, M

    1998-08-01

    Although chronic aluminum neurotoxicity has been well established, the mechanism of the toxicity has not been elucidated yet. In order to simplify the study of the aluminum neurotoxicity, we employed the pulse exposure of cultured rat cortical neurons to 250 microM aluminum-maltol for 1 h at the early stage (6 h after plating), which resulted in abnormal distribution of neurofilament L (NFL) and fast axonal transported proteins, whereas the axonal transport of tubulin, actin, and clathrin were not impaired. Otherwise, the pulse exposure of neurons at the late stage (4 days after plating) to the same concentration of aluminum-maltol did not affect the cell morphology and the distribution of NFL. The pulse exposure of cultured neurons to aluminum-maltol at the early stage might affect the axonal transport system of NFL and fast axonal transported proteins. PMID:9756345

  8. Peripheral axon crush elevates transport of p75NTR in the central projection of sensory neurones of rats.

    PubMed

    Delcroix, Jean-Dominique; Patel, Jyoti; Averill, Sharon; Tomlinson, David R; Priestley, John V; Fernyhough, Paul

    2003-11-20

    The effect of peripheral axon crush on the axonal transport of the neurotrophin receptors, p75(NTR) and trkA, was studied in dorsal roots of adult rats. Lumbar dorsal roots were crushed for 3-6 h to cause accumulation of p75(NTR) and trkA. Immunohistochemistry showed the presence of the NGF receptors in axons, indicating retrograde and anterograde axonal transport in the dorsal root. Western blots confirmed that the time course of accumulation of p75(NTR) was consistent with fast axonal transport. However, trkA accumulation was too low to indicate significant levels of axonal transport. Sciatic nerve crush induced a 2-fold increase (P<0.05) in the bidirectional axonal transport of p75(NTR) in the dorsal root while trkA transport remained below detectable levels. PMID:14623136

  9. A PIK3C3-ankyrin-B-dynactin pathway promotes axonal growth and multiorganelle transport.

    PubMed

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

    2014-12-22

    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

  10. Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

    PubMed Central

    Zhao, Xiaobei; Zhou, Yue; Weissmiller, April M.; Pearn, Matthew L.; Mobley, William C.; Wu, Chengbiao

    2014-01-01

    BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 μm/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders. PMID:25286194

  11. Kinesin-II Is Required for Axonal Transport of Choline Acetyltransferase in Drosophila

    PubMed Central

    Ray, Krishanu; Perez, Sharon E.; Yang, Zhaohuai; Xu, Jenny; Ritchings, Bruce W.; Steller, Hermann; Goldstein, Lawrence S.B.

    1999-01-01

    KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport. PMID:10545496

  12. A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice

    PubMed Central

    Kieran, Dairin; Hafezparast, Majid; Bohnert, Stephanie; Dick, James R.T.; Martin, Joanne; Schiavo, Giampietro; Fisher, Elizabeth M.C.; Greensmith, Linda

    2005-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for ∼20–25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1G93A mice during embryonic development. Surprisingly, crossing SOD1G93A mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1G93A mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS. PMID:15911875

  13. Absence of disturbed axonal transport in spinal and bulbar muscular atrophy

    PubMed Central

    Malik, Bilal; Nirmalananthan, Niranjanan; Bilsland, Lynsey G.; La Spada, Albert R.; Hanna, Michael G.; Schiavo, Giampietro; Gallo, Jean-Marc; Greensmith, Linda

    2011-01-01

    Spinal and bulbar muscular atrophy (SBMA), or Kennedy's disease, is a late-onset motor neuron disease (MND) caused by an abnormal expansion of the CAG repeat in the androgen receptor (AR) gene on the X-chromosome, encoding a polyglutamine (poly-Q) sequence in the protein product. Mutant poly-Q-expanded AR protein is widely expressed but leads to selective lower motoneuron death. Although the mechanisms that underlie SBMA remain unclear, defective axonal transport has been implicated in MND and other forms of poly-Q disease. Transcriptional dysregulation may also be involved in poly-Q repeat pathology. We therefore examined axonal transport in a mouse model of SBMA recapitulating many aspects of the human disease. We found no difference in the expression levels of motor and the microtubule-associated protein tau, in the spinal cord and sciatic nerve of wild-type (WT) and SBMA mice at various stages of disease progression. Furthermore, we found no alteration in binding properties of motor proteins and tau to microtubules. Moreover, analysis of axonal transport rates both in cultured primary motoneurons in vitro and in vivo in the sciatic nerve of adult WT and mutant SBMA mice demonstrated no overt axonal transport deficits in these systems. Our results therefore indicate that unlike other motoneuron and poly-Q diseases, axonal transport deficits do not play a significant role in the pathogenesis of SBMA. PMID:21317158

  14. 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.

  15. Cryo Electron Tomography of Herpes Simplex Virus during Axonal Transport and Secondary Envelopment in Primary Neurons

    PubMed Central

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

    2011-01-01

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

  16. Axonal transport of organelles visualized by light microscopy: cinemicrographic and computer analysis.

    PubMed

    Forman, D S; Padjen, A L; Siggins, G R

    1977-11-11

    Rapid movements of intra-axonal organelles in acutely isolated single myelinated fibers from bullfrog sciatic nerve were visualized by dark-field microscopy. The movements were recorded by cinemicrography, and analyzed by computer-based methods. The movements are saltatory and bidirectional, but each particle moves mainly in a single direction. For more than 90% of the particles, the predominant movement direction is retrograde, i.e. toward the cell body. Quantitative measurements on a variety of parameters of the organelle movements are presented. Different particles in the same axon show a broad range of mean speeds. The average mean speed of movement in the retrograde direction at 28 degrees C was 1.08 micrometer/sec (S.D. - 0.41), equivalent to an axonal transport rate of 93 mm/day. Disperse distributions were also found for other parameters such as the instantaneous velocities of individual particles. Quantal velocities, periodic movement patterns, and specific 'channels' were not detected. When the data from a population of particles is treated statistically, the average mean speed, the distribution of velocities, and other statistical parameters are found to be similar in different axons studied at the same temperature. Direct microscopical observation of axonal organelle movement is a technique which provides information about axonal transport which is different from and complementary to that obtained from enzyme accumulation of radioactive tracer methods. PMID:72584

  17. 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

  18. 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. PMID:27560175

  19. 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

  20. 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

  1. Early axonal loss accompanied by impaired endocytosis, abnormal axonal transport, and decreased microtubule stability occur in the model of Krabbe’s disease

    PubMed Central

    Teixeira, Carla Andreia; Miranda, Catarina Oliveira; Sousa, Vera Filipe; Santos, Telma Emanuela; Malheiro, Ana Rita; Solomon, Melani; Maegawa, Gustavo H.; Brites, Pedro; Sousa, Mónica Mendes

    2015-01-01

    In Krabbe’s Disease (KD), a leukodystrophy caused by β-galactosylceramidase deficiency, demyelination and a myelin-independent axonopathy contribute to the severe neuropathology. Beyond axonopathy, we show that in Twitcher mice, a model of KD, a decreased number of axons both in the PNS and CNS, and of neurons in dorsal root ganglia (DRG), occurred before the onset of demyelination. Despite the early axonal loss, and although in vitro Twitcher neurites degenerated over time, Twitcher DRG neurons displayed an initial neurite overgrowth and, following sciatic nerve injury, Twitcher axons were regeneration-competent, at a timepoint where axonopathy was already ongoing. Psychosine, the toxic substrate that accumulates in KD, induced lipid raft clustering. At the mechanistic level, TrkA recruitment to lipid rafts was dysregulated in Twitcher neurons, and defective activation of the ERK1/2 and AKT pathways was identified. Besides defective recruitment of signaling molecules to lipid rafts, the early steps of endocytosis and the transport of endocytic and synaptic vesicles were impaired in Twitcher DRG neurons. Defects in axonal transport, specifically in the retrograde component, correlated with decreased levels of dynein, abnormal levels of post-translational tubulin modifications and decreased microtubule stability. The identification of the axonal defects that precede demyelination in KD, together with the finding that Twitcher axons are regeneration-competent when axonopathy is already installed, open new windows of action to effectively correct the neuropathology that characterizes this disorder. PMID:24607884

  2. LOCALLY SYNTHESIZED PHOSPHATIDYCHOLINE, BUT NOT PROTEIN, UNDERGOES RAPID RETROGRADE AXONAL TRANSPORT IN THE RAT SCIATIC NERVE

    EPA Science Inventory

    Retrograde axonal transport of phosphatidylcholine (PC) in the sciatic nerve has been demonstrated only after injection of lipid precursors into the cell body regions (Armstrong et al. 1985). icroinjection of [methyl-3H]choline into the sciatic nerve results in extensive incorpor...

  3. 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.

  4. 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

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

    SciTech Connect

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

    1988-12-01

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

  6. Loss of the m-AAA protease subunit AFG₃L₂ causes mitochondrial transport defects and tau hyperphosphorylation.

    PubMed

    Kondadi, Arun Kumar; Wang, Shuaiyu; Montagner, Sara; Kladt, Nikolay; Korwitz, Anne; Martinelli, Paola; Herholz, David; Baker, Michael J; Schauss, Astrid C; Langer, Thomas; Rugarli, Elena I

    2014-05-01

    The m-AAA protease subunit AFG₃L₂ is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG₃L₂ are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG₃L₂ causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG₃L₂ is still unclear. Here, we show that depletion of AFG₃L₂ leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG₃L₂ in OMA1-deficient neurons, indicating that they are not caused by OMA1-mediated degradation of the dynamin-like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N-acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG₃L₂-depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for Afg3l2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG₃L₂. PMID:24681487

  7. Local translation and retrograde axonal transport of CREB regulates IL-6-induced nociceptive plasticity

    PubMed Central

    2014-01-01

    Transcriptional regulation of genes by cyclic AMP response element binding protein (CREB) is essential for the maintenance of long-term memory. Moreover, retrograde axonal trafficking of CREB in response to nerve growth factor (NGF) is critical for the survival of developing primary sensory neurons. We have previously demonstrated that hindpaw injection of interleukin-6 (IL-6) induces mechanical hypersensitivity and hyperalgesic priming that is prevented by the local injection of protein synthesis inhibitors. However, proteins that are locally synthesized that might lead to this effect have not been identified. We hypothesized that retrograde axonal trafficking of nascently synthesized CREB might link local, activity-dependent translation to nociceptive plasticity. To test this hypothesis, we determined if IL-6 enhances the expression of CREB and if it subsequently undergoes retrograde axonal transport. IL-6 treatment of sensory neurons in vitro caused an increase in CREB protein and in vivo treatment evoked an increase in CREB in the sciatic nerve consistent with retrograde transport. Importantly, co-injection of IL-6 with the methionine analogue azido-homoalanine (AHA), to assess nascently synthesized proteins, revealed an increase in CREB containing AHA in the sciatic nerve 2 hrs post injection, indicating retrograde transport of nascently synthesized CREB. Behaviorally, blockade of retrograde transport by disruption of microtubules or inhibition of dynein or intrathecal injection of cAMP response element (CRE) consensus sequence DNA oligonucleotides, which act as decoys for CREB DNA binding, prevented the development of IL-6-induced mechanical hypersensitivity and hyperalgesic priming. Consistent with previous studies in inflammatory models, intraplantar IL-6 enhanced the expression of BDNF in dorsal root ganglion (DRG). This effect was blocked by inhibition of retrograde axonal transport and by intrathecal CRE oligonucleotides. Collectively, these findings

  8. DRP1 inhibition rescues retinal ganglion cells and their axons by preserving mitochondrial integrity in a mouse model of glaucoma.

    PubMed

    Kim, K-Y; Perkins, G A; Shim, M S; Bushong, E; Alcasid, N; Ju, S; Ellisman, M H; Weinreb, R N; Ju, W-K

    2015-01-01

    Glaucoma is the leading cause of irreversible blindness and is characterized by slow and progressive degeneration of the optic nerve head axons and retinal ganglion cell (RGC), leading to loss of visual function. Although oxidative stress and/or alteration of mitochondrial (mt) dynamics induced by elevated intraocular pressure (IOP) are associated with this neurodegenerative disease, the mechanisms that regulate mt dysfunction-mediated glaucomatous neurodegeneration are poorly understood. Using a mouse model of glaucoma, DBA/2J (D2), which spontaneously develops elevated IOP, as well as an in vitro RGC culture system, we show here that oxidative stress, as evidenced by increasing superoxide dismutase 2 (SOD2) and mt transcription factor A (Tfam) protein expression, triggers mt fission and loss by increasing dynamin-related protein 1 (DRP1) in the retina of glaucomatous D2 mice as well as in cultured RGCs exposed to elevated hydrostatic pressure in vitro. DRP1 inhibition by overexpressing DRP1 K38A mutant blocks mt fission and triggers a subsequent reduction of oxidative stress, as evidenced by decreasing SOD2 and Tfam protein expression. DRP1 inhibition promotes RGC survival by increasing phosphorylation of Bad at serine 112 in the retina and preserves RGC axons by maintaining mt integrity in the glial lamina of glaucomatous D2 mice. These findings demonstrate an important vicious cycle involved in glaucomatous neurodegeneration that starts with elevated IOP producing oxidative stress; the oxidative stress then leads to mt fission and a specific form of mt dysfunction that generates further oxidative stress, thus perpetuating the cycle. Our findings suggest that DRP1 is a potential therapeutic target for ameliorating oxidative stress-mediated mt fission and dysfunction in RGC and its axons during glaucomatous neurodegeneration. Thus, DRP1 inhibition may provide a new therapeutic strategy for protecting both RGCs and their axons in glaucoma and other optic

  9. Teaching the Role of Mitochondrial Transport in Energy Metabolism

    ERIC Educational Resources Information Center

    Passarella, Salvatore; Atlante, Anna

    2007-01-01

    Studies from our laboratories over recent years have uncovered the existence, and established the properties of a variety of mitochondrial transporters. The properties of these transporters throw light on a variety of biochemical phenomena that were previously poorly understood. In particular the role of mitochondrial transport in energy…

  10. Analytical solution of equations describing slow axonal transport based on the stop-and-go hypothesis

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Andrey

    2011-06-01

    This paper presents an analytical solution for slow axonal transport in an axon. The governing equations for slow axonal transport are based on the stop-and-go hypothesis which assumes that organelles alternate between short periods of rapid movement on microtubules (MTs), short on-track pauses, and prolonged off-track pauses, when they temporarily disengage from MTs. The model includes six kinetic states for organelles: two for off-track organelles (anterograde and retrograde), two for running organelles, and two for pausing organelles. An analytical solution is obtained for a steady-state situation. To obtain the analytical solution, the governing equations are uncoupled by using a perturbation method. The solution is validated by comparing it with a high-accuracy numerical solution. Results are presented for neurofilaments (NFs), which are characterized by small diffusivity, and for tubulin oligomers, which are characterized by large diffusivity. The difference in transport modes between these two types of organelles in a short axon is discussed. A comparison between zero-order and first-order approximations makes it possible to obtain a physical insight into the effects of organelle reversals (when organelles change the type of a molecular motor they are attached to, an anterograde versus retrograde motor).

  11. 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. PMID:27517091

  12. 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

  13. 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

  14. 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

  15. Neurotrophic factors in Alzheimer’s disease: role of axonal transport

    PubMed Central

    Schindowski, K; Belarbi, K; Buée, L

    2008-01-01

    Neurotrophic factors (NTF) are small, versatile proteins that maintain survival and function to specific neuronal populations. In general, the axonal transport of NTF is important as not all of them are synthesized at the site of its action. Nerve growth factor (NGF), for instance, is produced in the neocortex and the hippocampus and then retrogradely transported to the cholinergic neurons of the basal forebrain. Neurodegenerative dementias like Alzheimer’s disease (AD) are linked to deficits in axonal transport. Furthermore, they are also associated with imbalanced distribution and dysregulation of NTF. In particular, brain-derived neurotrophic factor (BDNF) plays a crucial role in cognition, learning and memory formation by modulating synaptic plasticity and is, therefore, a critical molecule in dementia and neurodegenerative diseases. Here, we review the changes of NTF expression and distribution (NGF, BDNF, neurotrophin-3, neurotrophin-4/5 and fibroblast growth factor-2) and their receptors [tropomyosin-related kinase (Trk)A, TrkB, TrkC and p75NTR] in AD and AD models. In addition, we focus on the interaction with neuropathological hallmarks Tau/neurofibrillary tangle and amyloid-β (Abeta)/amyloid plaque pathology and their influence on axonal transport processes in order to unify AD-specific cholinergic degeneration and Tau and Abeta misfolding through NTF pathophysiology. PMID:18184369

  16. A fast and robust method for automated analysis of axonal transport.

    PubMed

    Welzel, Oliver; Knörr, Jutta; Stroebel, Armin M; Kornhuber, Johannes; Groemer, Teja W

    2011-09-01

    Cargo movement along axons and dendrites is indispensable for the survival and maintenance of neuronal networks. Key parameters of this transport such as particle velocities and pausing times are often studied using kymograph construction, which converts the transport along a line of interest from a time-lapse movie into a position versus time image. Here we present a method for the automatic analysis of such kymographs based on the Hough transform, which is a robust and fast technique to extract lines from images. The applicability of the method was tested on simulated kymograph images and real data from axonal transport of synaptophysin and tetanus toxin as well as the velocity analysis of synaptic vesicle sharing between adjacent synapses in hippocampal neurons. Efficiency analysis revealed that the algorithm is able to detect a wide range of velocities and can be used at low signal-to-noise ratios. The present work enables the quantification of axonal transport parameters with high throughput with no a priori assumptions and minimal human intervention. PMID:21695534

  17. Hepatic mitochondrial glutathione: transport and role in disease and toxicity

    SciTech Connect

    Fernandez-Checa, Jose C. . E-mail: checa229@yahoo.com; Kaplowitz, Neil . E-mail: kaplowitz@hsc.usc.edu

    2005-05-01

    Synthesized in the cytosol of cells, a fraction of cytosolic glutathione (GSH) is then transported into the mitochondrial matrix where it reaches a high concentration and plays a critical role in defending mitochondria against oxidants and electrophiles. Evidence mainly from kidney and liver mitochondria indicated that the dicarboxylate and the 2-oxoglutarate carriers contribute to the transport of GSH across the mitochondrial inner membrane. However, differential features between kidney and liver mitochondrial GSH (mGSH) transport seem to suggest the existence of additional carriers the identity of which remains to be established. One of the characteristic features of the hepatic mitochondrial transport of GSH is its regulation by membrane fluidity. Conditions leading to increased cholesterol deposition in the mitochondrial inner membrane such as in alcohol-induced liver injury decrease membrane fluidity and impair the mitochondrial transport of GSH. Depletion of mitochondrial GSH by alcohol is believed to contribute to the sensitization of the liver to alcohol-induced injury through tumor necrosis factor (TNF)-mediated hepatocellular death. Through control of mitochondrial electron transport chain-generated oxidants, mitochondrial GSH modulates cell death and hence its regulation may be a key target to influence disease progression and drug-induced cell death.

  18. Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons.

    PubMed

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

    2014-01-27

    Action potentials (APs) propagating along axons require the activation of voltage-gated Na(+) (Nav) channels. How Nav channels are transported into axons is unknown. We show that 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 small interfering RNA or dominant-negative constructs markedly reduced Nav channel levels at the axon initial segment (AIS) and along entire axons, thereby decreasing AP firing. Live-cell imaging showed that fluorescently tagged AnkG or Nav1.2 cotransported 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 mouse cerebellum. These results indicate that 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

  19. 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

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

    SciTech Connect

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

    1992-01-01

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

  1. Demyelination induces transport of ribosome-containing vesicles from glia to axons: evidence from animal models and MS patient brains.

    PubMed

    Shakhbazau, Antos; Schenk, Geert J; Hay, Curtis; Kawasoe, Jean; Klaver, Roel; Yong, V Wee; Geurts, Jeroen J G; van Minnen, Jan

    2016-06-01

    Glial cells were previously proven capable of trafficking polyribosomes to injured axons. However, the occurrence of such transfer in the general pathological context, such as demyelination-related diseases, needs further evidence. Since this may be a yet unidentified universal contributor to axonal survival, we study putative glia-axonal ribosome transport in response to demyelination in animal models and patients in both peripheral and central nervous system. In the PNS we investigate whether demyelination in a rodent model has the potential to induce ribosome transfer. We also probe the glia-axonal ribosome supply by implantation of transgenic Schwann cells engineered to produce fluorescent ribosomes in the same demyelination model. We furthermore examine the presence of axonal ribosomes in mouse experimental autoimmune encephalomyelitis (EAE), a well-established model for multiple sclerosis (MS), and in human MS autopsy brain material. We provide evidence for increased axonal ribosome content in a pharmacologically demyelinated sciatic nerve, and demonstrate that at least part of these ribosomes originate in the transgenic Schwann cells. In the CNS one of the hallmarks of MS is demyelination, which is associated with severe disruption of oligodendrocyte-axon interaction. Here, we provide evidence that axons from spinal cords of EAE mice, and in the MS human brain contain an elevated amount of axonal ribosomes compared to controls. Our data provide evidence that increased axonal ribosome content in pathological axons is at least partly due to glia-to-axon transfer of ribosomes, and that demyelination in the PNS and in the CNS is one of the triggers capable to initiate this process. PMID:27115494

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

    PubMed

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

    2016-05-01

    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

  3. 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

  4. 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

  5. The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport

    PubMed Central

    Araki, Yoichi; Kawano, Takanori; Taru, Hidenori; Saito, Yuhki; Wada, Sachiyo; Miyamoto, Kanako; Kobayashi, Hisako; Ishikawa, Hiroyuki O; Ohsugi, Yu; Yamamoto, Tohru; Matsuno, Kenji; Kinjo, Masataka; Suzuki, Toshiharu

    2007-01-01

    Alcadeinα (Alcα) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alcα strongly associates with kinesin light chain (KD≈4–8 × 10−9 M) through a novel tryptophan- and aspartic acid-containing sequence. Alcα can induce kinesin-1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK-interacting protein 1 (JIP1), an adaptor protein for kinesin-1, perturbs the transport of Alcα, and the kinesin-1 motor complex dissociates from Alcα-containing vesicles in a JIP1 concentration-dependent manner. Alcα-containing vesicles were transported with a velocity different from that of amyloid β-protein precursor (APP)-containing vesicles, which are transported by the same kinesin-1 motor. Alcα- and APP-containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alcα with kinesin-1 blocked transport of APP-containing vesicles and increased β-amyloid generation. Inappropriate interactions of Alc- and APP-containing vesicles with kinesin-1 may promote aberrant APP metabolism in Alzheimer's disease. PMID:17332754

  6. 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. PMID:11891785

  7. Formation of α-synuclein Lewy neurite–like aggregates in axons impedes the transport of distinct endosomes

    PubMed Central

    Volpicelli-Daley, Laura A.; Gamble, Karen L.; Schultheiss, Christine E.; Riddle, Dawn M.; West, Andrew B.; Lee, Virginia M.-Y.

    2014-01-01

    Aggregates of α-synuclein (α-syn) accumulate in neurons in Parkinson's disease and other synucleinopathies. These inclusions predominantly localize to axons even in the early stages of the disease, but their affect on axon function has remained unknown. Previously we established a model in which the addition of preformed α-syn fibrils to primary neurons seeds formation of insoluble α-syn inclusions built from endogenously expressed α-syn that closely recapitulate the neuropathological phenotypes of Lewy neurites found in human diseased brains. Here we show, using live-cell imaging, that immobile α-syn inclusions accumulate in axons from the recruitment of α-syn located on mobile α-syn–positive vesicles. Ultrastructural analyses and live imaging demonstrate that α-syn accumulations do not cause a generalized defect in axonal transport; the inclusions do not fill the axonal cytoplasm, disrupt the microtubule cytoskeleton, or affect the transport of synaptophysin or mitochondria. However, the α-syn aggregates impair the transport of Rab7 and TrkB receptor–containing endosomes, as well as autophagosomes. In addition, the TrkB receptor–associated signaling molecule pERK5 accumulates in α-syn aggregate–bearing neurons. Thus α-syn pathology impairs axonal transport of signaling and degradative organelles. These early effects of α-syn accumulations may predict points of intervention in the neurodegenerative process. PMID:25298402

  8. Identification of mitochondrial coenzyme a transporters from maize and Arabidopsis.

    PubMed

    Zallot, Rémi; Agrimi, Gennaro; Lerma-Ortiz, Claudia; Teresinski, Howard J; Frelin, Océane; Ellens, Kenneth W; Castegna, Alessandra; Russo, Annamaria; de Crécy-Lagard, Valérie; Mullen, Robert T; Palmieri, Ferdinando; Hanson, Andrew D

    2013-06-01

    Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes. PMID:23590975

  9. Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration

    PubMed Central

    Sheng, Zu-Hang; Cai, Qian

    2016-01-01

    Mitochondria have a number of essential roles in neuronal function. Their complex mobility patterns within neurons are characterized by frequent changes in direction. Mobile mitochondria can become stationary or pause in regions that have a high metabolic demand and can move again rapidly in response to physiological changes. Defects in mitochondrial transport are implicated in the pathogenesis of several major neurological disorders. Research into the mechanisms that regulate mitochondrial transport is thus an important emerging frontier. PMID:22218207

  10. Mitochondrial pyruvate transport: a historical perspective and future research directions

    PubMed Central

    McCommis, Kyle S.; Finck, Brian N.

    2015-01-01

    Pyruvate is the end-product of glycolysis, a major substrate for oxidative metabolism, and a branching point for glucose, lactate, fatty acid and amino acid synthesis. The mitochondrial enzymes that metabolize pyruvate are physically separated from cytosolic pyruvate pools and rely on a membrane transport system to shuttle pyruvate across the impermeable inner mitochondrial membrane (IMM). Despite long-standing acceptance that transport of pyruvate into the mitochondrial matrix by a carrier-mediated process is required for the bulk of its metabolism, it has taken almost 40 years to determine the molecular identity of an IMM pyruvate carrier. Our current understanding is that two proteins, mitochondrial pyruvate carriers MPC1 and MPC2, form a hetero-oligomeric complex in the IMM to facilitate pyruvate transport. This step is required for mitochondrial pyruvate oxidation and carboxylation – critical reactions in intermediary metabolism that are dysregulated in several common diseases. The identification of these transporter constituents opens the door to the identification of novel compounds that modulate MPC activity, with potential utility for treating diabetes, cardiovascular disease, cancer, neurodegenerative diseases, and other common causes of morbidity and mortality. The purpose of the present review is to detail the historical, current and future research investigations concerning mitochondrial pyruvate transport, and discuss the possible consequences of altered pyruvate transport in various metabolic tissues. PMID:25748677

  11. Cytoprotection by the Modulation of Mitochondrial Electron Transport Chain: The Emerging Role of Mitochondrial STAT3

    PubMed Central

    Szczepanek, Karol; Chen, Qun; Larner, Andrew C.; Lesnefsky, Edward J.

    2011-01-01

    The down regulation of mitochondrial electron transport is an emerging mechanism of cytoprotective intervention that is effective in pathologic settings such as myocardial ischemia and reperfusion when the continuation of mitochondrial respiration produces reactive oxygen species, mitochondrial calcium overload, and the release of cytochrome c to activate cell death programs. The initial target of deranged electron transport is the mitochondria themselves. In the first part of this review, we describe this concept and summarize different approaches used to regulate mitochondrial respiration by targeting complex I as a proximal site in the electron transport chain (ETC) in order to favor the cytoprotection. The second part of the review highlights the emerging role of signal transducer and activator of transcription 3 (STAT3) in the direct, non-transcriptional regulation of ETC, as an example of a genetic approach to modulate respiration. Recent studies indicate that a pool of STAT3 resides in the mitochondria where it is necessary for the maximal activity of complexes I and II of the electron transport chain (ETC). The over expression of mitochondrial-targeted STAT3 results in a partial blockade of electron transport at complexes I and II that does not impair mitochondrial membrane potential nor enhance the production of reactive oxygen species (ROS). The targeting of transcriptionally-inactive STAT3 to mitochondria attenuates damage to mitochondria during cell stress, resulting in decreased production of ROS and retention of cytochrome c by mitochondria. The overexpression of STAT3 targeted to mitochondria unveils a novel protective approach mediated by modulation of mitochondrial respiration that is independent of STAT3 transcriptional activity. The limitation of mitochondrial respiration under pathologic circumstances can be approached by activation and over expression of endogenous signaling mechanisms in addition to pharmacologic means. The regulation of

  12. Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia

    PubMed Central

    Ittner, Lars M.; Fath, Thomas; Ke, Yazi D.; Bi, Mian; van Eersel, Janet; Li, Kong M.; Gunning, Peter; Götz, Jürgen

    2008-01-01

    Frontotemporal dementia (FTD) is characterized by cognitive and behavioral changes and, in a significant subset of patients, Parkinsonism. Histopathologically, FTD frequently presents with tau-containing lesions, which in familial cases result from mutations in the MAPT gene encoding tau. Here we present a novel transgenic mouse strain (K3) that expresses human tau carrying the FTD mutation K369I. K3 mice develop a progressive histopathology that is reminiscent of that in human FTD with the K369I mutation. In addition, K3 mice show early-onset memory impairment and amyotrophy in the absence of overt neurodegeneration. Different from our previously generated tau transgenic strains, the K3 mice express the transgene in the substantia nigra (SN) and show an early-onset motor phenotype that reproduces Parkinsonism with tremor, bradykinesia, abnormal gait, and postural instability. Interestingly, motor performance of young, but not old, K3 mice improves upon L-dopa treatment, which bears similarities to Parkinsonism in FTD. The early-onset symptoms in the K3 mice are mechanistically related to selectively impaired anterograde axonal transport of distinct cargos, which precedes the loss of dopaminergic SN neurons that occurs in aged mice. The impaired axonal transport in SN neurons affects, among others, vesicles containing the dopamine-synthesizing enzyme tyrosine hydroxylase. Distinct modes of transport are also impaired in sciatic nerves, which may explain amyotrophy. Together, the K3 mice are a unique model of FTD-associated Parkinsonism, with pathomechanistic implications for the human pathologic process. PMID:18832465

  13. 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

    2015-01-01

    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. PMID:26586091

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:26586091

  15. Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles

    PubMed Central

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

    2013-01-01

    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. PMID:19787448

  16. Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics*

    PubMed Central

    Wei, An-Chi; Liu, Ting; O'Rourke, Brian

    2015-01-01

    The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca2+ uptake, these include activation of countertransporters (Na+/Ca2+ exchanger and Na+/H+ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca2+ binding to matrix buffers. Inorganic phosphate (Pi) is known to affect both the Ca2+ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca2+ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca2+ and mitochondrial membrane potential (ΔΨm) to examine the effects of anion transport on mitochondrial Ca2+ flux and buffering in Pi-depleted guinea pig cardiac mitochondria. Mitochondrial Ca2+ uptake proceeded slowly in the absence of Pi but matrix free Ca2+ ([Ca2+]mito) still rose to ∼50 μm. Pi (0.001–1 mm) accelerated Ca2+ uptake but decreased [Ca2+]mito by almost 50% while restoring ΔΨm. Pi-dependent effects on Ca2+ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca2+ uptake rate was also increased by vanadate (Vi), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca2+ buffering and ΔΨm recovery. Interestingly, ATP or AMP-PNP prevented the effects of Pi on Ca2+ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca2+ uptake and modifies the [Ca2+]mito response in a complex manner. PMID:25963147

  17. 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.

  18. Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta

    PubMed Central

    Pigino, G.; Morfini, G.; Atagi, Y.; Deshpande, A.; Yu, C.; Jungbauer, L.; LaDu, M.; Busciglio, J.; Brady, S.

    2009-01-01

    The pathological mechanism by which Aβ causes neuronal dysfunction and death remains largely unknown. Deficiencies in fast axonal transport (FAT) were suggested to play a crucial role in neuronal dysfunction and loss for a diverse set of dying back neuropathologies including Alzheimer's disease (AD), but the molecular basis for pathological changes in FAT were undetermined. Recent findings indicate that soluble intracellular oligomeric Aβ (oAβ) species may play a critical role in AD pathology. Real-time analysis of vesicle mobility in isolated axoplasms perfused with oAβ showed bidirectional axonal transport inhibition as a consequence of endogenous casein kinase 2 (CK2) activation. Conversely, neither unaggregated amyloid beta nor fibrillar amyloid beta affected FAT. Inhibition of FAT by oAβ was prevented by two specific pharmacological inhibitors of CK2, as well as by competition with a CK2 substrate peptide. Furthermore, perfusion of axoplasms with active CK2 mimics the inhibitory effects of oAβ on FAT. Both oAβ and CK2 treatment of axoplasm led to increased phosphorylation of kinesin-1 light chains and subsequent release of kinesin from its cargoes. Therefore pharmacological modulation of CK2 activity may represent a promising target for therapeutic intervention in AD. PMID:19321417

  19. Automated measurement of fast mitochondrial transport in neurons

    PubMed Central

    Miller, Kyle E.; Liu, Xin-An; Puthanveettil, Sathyanarayanan V.

    2015-01-01

    There is growing recognition that fast mitochondrial transport in neurons is disrupted in multiple neurological diseases and psychiatric disorders. However, a major constraint in identifying novel therapeutics based on mitochondrial transport is that the large-scale analysis of fast transport is time consuming. Here we describe methodologies for the automated analysis of fast mitochondrial transport from data acquired using a robotic microscope. We focused on addressing questions of measurement precision, speed, reliably, workflow ease, statistical processing, and presentation. We used optical flow and particle tracking algorithms, implemented in ImageJ, to measure mitochondrial movement in primary cultured cortical and hippocampal neurons. With it, we are able to generate complete descriptions of movement profiles in an automated fashion of hundreds of thousands of mitochondria with a processing time of approximately one hour. We describe the calibration of the parameters of the tracking algorithms and demonstrate that they are capable of measuring the fast transport of a single mitochondrion. We then show that the methods are capable of reliably measuring the inhibition of fast mitochondria transport induced by the disruption of microtubules with the drug nocodazole in both hippocampal and cortical neurons. This work lays the foundation for future large-scale screens designed to identify compounds that modulate mitochondrial motility. PMID:26578890

  20. Automated measurement of fast mitochondrial transport in neurons.

    PubMed

    Miller, Kyle E; Liu, Xin-An; Puthanveettil, Sathyanarayanan V

    2015-01-01

    There is growing recognition that fast mitochondrial transport in neurons is disrupted in multiple neurological diseases and psychiatric disorders. However, a major constraint in identifying novel therapeutics based on mitochondrial transport is that the large-scale analysis of fast transport is time consuming. Here we describe methodologies for the automated analysis of fast mitochondrial transport from data acquired using a robotic microscope. We focused on addressing questions of measurement precision, speed, reliably, workflow ease, statistical processing, and presentation. We used optical flow and particle tracking algorithms, implemented in ImageJ, to measure mitochondrial movement in primary cultured cortical and hippocampal neurons. With it, we are able to generate complete descriptions of movement profiles in an automated fashion of hundreds of thousands of mitochondria with a processing time of approximately one hour. We describe the calibration of the parameters of the tracking algorithms and demonstrate that they are capable of measuring the fast transport of a single mitochondrion. We then show that the methods are capable of reliably measuring the inhibition of fast mitochondria transport induced by the disruption of microtubules with the drug nocodazole in both hippocampal and cortical neurons. This work lays the foundation for future large-scale screens designed to identify compounds that modulate mitochondrial motility. PMID:26578890

  1. Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-beta-hydroxylase.

    PubMed

    Laduron, P M

    1984-01-01

    Presynaptic muscarinic receptors labeled with [3H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [3H]quinuclidinyl benzilate or [3H]dexetimide binding revealed a distribution profile similar to that of dopamine-beta-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-beta-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-beta-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle. PMID:6198205

  2. 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

  3. A biophysical analysis of mitochondrial movement: differences between transport in neuronal cell bodies versus processes.

    PubMed

    Narayanareddy, Babu Reddy Janakaloti; Vartiainen, Suvi; Hariri, Neema; O'Dowd, Diane K; Gross, Steven P

    2014-07-01

    There is an increasing interest in factors that can impede cargo transport by molecular motors inside the cell. Although potentially relevant (Yi JY, Ori-McKenney KM, McKenney RJ, Vershinin M, Gross SP, Vallee RB. High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport. J Cell Biol 2011;195:193-201), the importance of cargo size and subcellular location has received relatively little attention. Here we address these questions taking advantage of the fact that mitochondria - a common cargo - in Drosophila neurons exhibit a wide distribution of sizes. In addition, the mitochondria can be genetically marked with green fluorescent protein (GFP) making it possible to visualize and compare their movement in the cell bodies and in the processes of living cells. Using total internal reflection microscopy coupled with particle tracking and analysis, we quantified the transport properties of GFP-positive mitochondria as a function of their size and location. In neuronal cell bodies, we find little evidence for significant opposition to motion, consistent with a previous study on lipid droplets (Shubeita GT, Tran SL, Xu J, Vershinin M, Cermelli S, Cotton SL, Welte MA, Gross SP. Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets. Cell 2008;135:1098-1107). However, in the processes, we observe an inverse relationship between the mitochondrial size and velocity and the run distances. This can be ameliorated via hypotonic treatment to increase process size, suggesting that motor-mediated movement is impeded in this more-confined environment. Interestingly, we also observe local mitochondrial accumulations in processes but not in cell bodies. Such accumulations do not completely block the transport but do increase the probability of mitochondria-mitochondria interactions. They are thus particularly interesting in relation to mitochondrial exchange of elements

  4. 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

  5. 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

  6. Nanoparticle-assisted optical tethering of endosomes reveals the cooperative function of dyneins in retrograde axonal transport

    PubMed Central

    Chowdary, Praveen D.; Che, Daphne L.; Kaplan, Luke; Chen, Ou; Pu, Kanyi; Bawendi, Moungi; Cui, Bianxiao

    2015-01-01

    Dynein-dependent transport of organelles from the axon terminals to the cell bodies is essential to the survival and function of neurons. However, quantitative knowledge of dyneins on axonal organelles and their collective function during this long-distance transport is lacking because current technologies to do such measurements are not applicable to neurons. Here, we report a new method termed nanoparticle-assisted optical tethering of endosomes (NOTE) that made it possible to study the cooperative mechanics of dyneins on retrograde axonal endosomes in live neurons. In this method, the opposing force from an elastic tether causes the endosomes to gradually stall under load and detach with a recoil velocity proportional to the dynein forces. These recoil velocities reveal that the axonal endosomes, despite their small size, can recruit up to 7 dyneins that function as independent mechanical units stochastically sharing load, which is vital for robust retrograde axonal transport. This study shows that NOTE, which relies on controlled generation of reactive oxygen species, is a viable method to manipulate small cellular cargos that are beyond the reach of current technology. PMID:26656461

  7. Is abnormal axonal transport a cause, a contributing factor or a consequence of the neuronal pathology in Alzheimer’s disease?

    PubMed Central

    Muresan, Virgil; Muresan, Zoia

    2009-01-01

    Axonal transport, the process by which membrane-bound organelles and soluble protein complexes are transported into and out of axons, ensures proper function of the neuron, including that of the synapse. As such, abnormalities in axonal transport could lead to neuronal pathology and disease. Similar to many neurodegenerative diseases, axonal transport is deficient in Alzheimer’s disease (AD), a neurodegenerative brain disorder that affects old-age humans and is characterized by the deterioration of cognitive function and progressive memory loss. It was proposed that the synaptic pathology and neuronal degeneration that develops in AD could be caused by an abnormal axonal transport, and that the mutated proteins that cause early-onset AD, as well as the genetic variants that confer predisposition to late-onset AD might somehow impede axonal transport. This paper analyzes the data that support or contradict this hypothesis. Together, they indicate that, although abnormalities in axonal transport are part of the disease, additional studies are required to clearly establish to what extent deficient axonal transport is the cause or the effect of the neuronal pathology in AD, and to identify mechanisms that lead to its perturbation. PMID:20076770

  8. Mitochondrial transporters for ornithine and related amino acids: a review.

    PubMed

    Monné, Magnus; Miniero, Daniela Valeria; Daddabbo, Lucia; Palmieri, Luigi; Porcelli, Vito; Palmieri, Ferdinando

    2015-09-01

    Among the members of the mitochondrial carrier family, there are transporters that catalyze the translocation of ornithine and related substrates, such as arginine, homoarginine, lysine, histidine, and citrulline, across the inner mitochondrial membrane. The mitochondrial carriers ORC1, ORC2, and SLC25A29 from Homo sapiens, BAC1 and BAC2 from Arabidopsis thaliana, and Ort1p from Saccharomyces cerevisiae have been biochemically characterized by transport assays in liposomes. All of them transport ornithine and amino acids with side chains terminating at least with one amine. There are, however, marked differences in their substrate specificities including their affinity for ornithine (KM values in the mM to μM range). These differences are most likely reflected by minor differences in the substrate binding sites of these carriers. The physiological role of the above-mentioned mitochondrial carriers is to link several metabolic pathways that take place partly in the cytosol and partly in the mitochondrial matrix and to provide basic amino acids for mitochondrial translation. In the liver, human ORC1 catalyzes the citrulline/ornithine exchange across the mitochondrial inner membrane, which is required for the urea cycle. Human ORC1, ORC2, and SLC25A29 are likely to be involved in the biosynthesis and transport of arginine, which can be used as a precursor for the synthesis of NO, agmatine, polyamines, creatine, glutamine, glutamate, and proline, as well as in the degradation of basic amino acids. BAC1 and BAC2 are implicated in some processes similar to those of their human counterparts and in nitrogen and amino acid metabolism linked to stress conditions and the development of plants. Ort1p is involved in the biosynthesis of arginine and polyamines in yeast. PMID:26002808

  9. Dual effect of spermine on mitochondrial Ca2+ transport.

    PubMed Central

    Lenzen, S; Münster, W; Rustenbeck, I

    1992-01-01

    1. A dual effect of the polyamine spermine on Ca2+ uptake by isolated rat liver, brain and heart mitochondria could be demonstrated by using a high-resolution system for studying mitochondrial Ca2+ transport. Depending on the experimental situation, spermine had an inhibiting or accelerating effects on mitochondrial Ca(2+)-uptake rate, but invariably increased the mitochondrial Ca2+ accumulation. 2. Both effects were concentration-dependent and clearly discernible on the basis of their different kinetic characteristics. For mitochondria from all three tissues the half-maximally effective concentration for inhibition of the initial rate of Ca2+ uptake was approx. 180 microM, whereas that for the subsequent stimulation of Ca2+ accumulation was approx. 50 microM. 3. Acceleration of the initial uptake rate could be seen when the mitochondria were preloaded with spermine during a 2 min preincubation period and thereafter incubated in a medium without spermine. 4. When such spermine-preloaded mitochondria were incubated in a spermine-containing medium, the increase in Ca(2+)-accumulation capacity was maintained in spite of an unchanged rate of Ca2+ uptake. 5. Mg2+ interacted with the effects of spermine in a differential manner, enhancing the initial inhibition of the rate of mitochondrial Ca2+ uptake and diminishing the subsequent stimulation of mitochondrial Ca2+ accumulation. 6. This dual effect of spermine on mitochondrial Ca2+ transport resolves the apparent paradox that a polycationic compound can act as a stimulator of Ca2+ uptake. PMID:1530590

  10. Stable Kinesin and Dynein Assemblies Drive the Axonal Transport of Mammalian Prion Protein Vesicles

    PubMed Central

    Encalada, Sandra E.; Szpankowski, Lukasz; Xia, Chun-hong; Goldstein, Lawrence S. B.

    2012-01-01

    SUMMARY Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo but their overall composition on axonal vesicles and whether this composition directly modulates transport activity, is unknown. Here we characterize the intracellular transport and steady state motor subunit composition of mammalian prion protein (PrPC) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrPC vesicle motor complexes, and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment, and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model where PrPC vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations. PMID:21335237

  11. Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations

    PubMed Central

    Godena, Vinay K.; Brookes-Hocking, Nicholas; Moller, Annekathrin; Shaw, Gary; Oswald, Matthew; Sancho, Rosa M.; Miller, Christopher C. J.; Whitworth, Alexander J.; De Vos, Kurt J.

    2014-01-01

    Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson’s disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson’s disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson’s disease. PMID:25316291

  12. Acrylamide Retards the Slow Axonal Transport of Neurofilaments in Rat Cultured Dorsal Root Ganglia Neurons and the Corresponding Mechanisms.

    PubMed

    An, Lihong; Li, Guozhen; Si, Jiliang; Zhang, Cuili; Han, Xiaoying; Wang, Shuo; Jiang, Lulu; Xie, Keqin

    2016-05-01

    Chronic acrylamide (ACR) exposure induces peripheral-central axonopathy in occupational workers and laboratory animals, but the underlying mechanisms remain unclear. In this study, we first investigated the effects of ACR on slow axonal transport of neurofilaments in cultured rat dorsal root ganglia (DRG) neurons through live-cell imaging approach. Then for the underlying mechanisms exploration, the protein level of neurofilament subunits, motor proteins kinesin and dynein, and dynamitin subunit of dynactin in DRG neurons were assessed by western blotting and the concentrations of ATP was detected using ATP Assay Kit. The results showed that ACR treatment results in a dose-dependent decrease of slow axonal transport of neurofilaments. Furthermore, ACR intoxication significantly increases the protein levels of the three neurofilament subunits (NF-L, NF-M, NF-H), kinesin, dynein, and dynamitin subunit of dynactin in DRG neurons. In addition, ATP level decreased significantly in ACR-treated DRG neurons. Our findings indicate that ACR exposure retards slow axonal transport of NF-M, and suggest that the increase of neurofilament cargoes, motor proteins, dynamitin of dynactin, and the inadequate ATP supply contribute to the ACR-induced retardation of slow axonal transport. PMID:26721510

  13. EFFECTS OF HYPOTHERMIA ON THE IN VIVO MEASUREMENT OF RAPID AXONAL TRANSPORT IN THE RAT: A CAUTIONARY NOTE

    EPA Science Inventory

    Rapid axonal transport of glycoproteins was examined in the retinofugal projections of hypothermic and normothermic adult male Long-Evans hooded rats previously receiving intraocular injections of (3H)fucose. The amount of retinal fucosylation appeared normal in the hypothermic a...

  14. Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport

    PubMed Central

    Shi, Ping; Ström, Anna-Lena; Gal, Jozsef; Zhu, Haining

    2010-01-01

    Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R and G93A) interacted and co-localized with the retrograde dynein-dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early pre-symptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the anterograde transport of dynein heavy chain as a cargo was observed in the pre-symptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members which in turn could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affect different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which subsequently contribute to the propagation of toxic effects and ultimately motor neuron death in ALS. PMID:20510358

  15. Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

    SciTech Connect

    Chen, Qun; Yin, Guotian; Stewart, Sarah; Hu, Ying; Lesnefsky, Edward J.

    2010-07-09

    Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 {sup o}C. Amobarbital (2.5 mM) or azide (5 mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.

  16. The dynamics and location of axonal transport blockade by acute intraocular pressure elevation in primate optic nerve.

    PubMed

    Quigley, H; Anderson, D R

    1976-08-01

    Axonal transport in primate optic nerve axons was studied by autoradiography, scintillation counting, and electron microscopy under conditions of short-term intraocular pressure elevation. With elevation of intraocular pressure to 30 mm. Hg below mean arterial blood pressure, blockage of transport was detected within 2 hours by autoradiography and within 1 hour by electron microscopy. The earliest buildup of radioactively labeled protein and ultrastructurally visible cellular organelles was within the scleral lamina cribrosa. The degree of blockage, judged by amount of label buildup, increased with time. Some transported material traversed the lamina cribrosa despite pressure elevation at the level tested. Reversal of transport blockade occurred rapidly after normalization of intraocular pressure. PMID:60300

  17. The transport mechanism of the mitochondrial ADP/ATP carrier.

    PubMed

    Kunji, Edmund R S; Aleksandrova, Antoniya; King, Martin S; Majd, Homa; Ashton, Valerie L; Cerson, Elizabeth; Springett, Roger; Kibalchenko, Mikhail; Tavoulari, Sotiria; Crichton, Paul G; Ruprecht, Jonathan J

    2016-10-01

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. PMID:27001633

  18. 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

  19. 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

  20. 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.

  1. Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

    PubMed Central

    Ryu, Shin-Young; Jhun, Bong Sook; Hurst, Stephen

    2014-01-01

    Abstract Significance: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. Recent Advances: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. Critical Issues: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. Future Directions: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters. Antioxid. Redox Signal. 21, 987–1006. PMID:24180309

  2. BODY TEMPERATURE-DEPENDENT AND INDEPENDENT ACTIONS OF CHLORDIMEFORM ON VISUAL EVOKED POTENTIALS AND AXONAL TRANSPORT IN OPTIC SYSTEM OF RAT

    EPA Science Inventory

    Pattern reversal evoked potentials (PREPs), flash evoked potentials (FEPs), optic nerve axonal transport, and body temperature were measured in hooded rats treated with either saline or the formamidine insecticide/acaricide, chlordimeform (CDM). Rats receiving CDM had low body te...

  3. Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells.

    PubMed

    Fukumitsu, Kansai; Hatsukano, Tetsu; Yoshimura, Azumi; Heuser, John; Fujishima, Kazuto; Kengaku, Mineko

    2016-03-01

    Mitochondria dynamically change their shape by repeated fission and fusion in response to physiological and pathological conditions. Recent studies have uncovered significant roles of mitochondrial fission and fusion in neuronal functions, such as neurotransmission and spine formation. However, the contribution of mitochondrial fission to the development of dendrites remains controversial. We analyzed the function of the mitochondrial fission GTPase Drp1 in dendritic arborization in cerebellar Purkinje cells. Overexpression of a dominant-negative mutant of Drp1 in postmitotic Purkinje cells enlarged and clustered mitochondria, which failed to exit from the soma into the dendrites. The emerging dendrites lacking mitochondrial transport remained short and unstable in culture and in vivo. The dominant-negative Drp1 affected neither the basal respiratory function of mitochondria nor the survival of Purkinje cells. Enhanced ATP supply by creatine treatment, but not reduced ROS production by antioxidant treatment, restored the hypomorphic dendrites caused by inhibition of Drp1 function. Collectively, our results suggest that Drp1 is required for dendritic distribution of mitochondria and thereby regulates energy supply in growing dendritic branches in developing Purkinje cells. PMID:26689905

  4. Cellular synthesis and axonal transport of gamma-aminobutyric acid in a photoreceptor cell of the barnacle.

    PubMed Central

    Koike, H; Tsuda, K

    1980-01-01

    1. [3H]glutamate or [3H]gamma-aminobutyric acid (GABA) was injected into the photoreceptor cell of the lateral ocellus of Balanus eburneus, in order to study the transmitter substance of the cell. 2. The photoreceptor cell synthesized [3H]GABA from injected [3H]glutamate. 3. The newly formed [3H]GABA moved inside the photoreceptor axon towards the axon terminal with a velocity of about 0.9 mm/hr. Injected [3H]GABA moved at 0.9 mm/hr and also at 0.4 mm/hr. 4. Axonally transported [3H]GABA reached the axon terminal within several hours following the injection. It did not accumulate at the terminal, but gradually disappeared. 5. Light-microscope and electron-microscope autoradiography following the injection of [3H]GABA revealed that [3H]-reacted silver grains were present in a certain type of axon terminal. The terminal thus identified as that of a photoreceptor cell contains many clear, polymorphic synaptic vesicles about 300-500 A in diameter, some dense-cored vesicles 700-1300 A in diameter, and glycogen granules. The terminal forms many synapses, and each synapse has a synaptic dense body. The terminal always faces two post-synaptic elements at the synapse, forming a triad with a gap distance of about 160-200 A. 6. A GABA analogue, [3H]di-aminobutyric acid, was selectively taken up into the terminals previously identified as those of photoreceptors. 7. These results support the notion that the transmitter substance of the photoreceptor cell of the barnacle is GABA. Images Plate 1 Plate 2 PMID:6160239

  5. 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

  6. 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.

  7. The RNA binding and transport proteins staufen and fragile X mental retardation protein are expressed by rat primary afferent neurons and localize to peripheral and central axons.

    PubMed

    Price, T J; Flores, C M; Cervero, F; Hargreaves, K M

    2006-09-15

    Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile x mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation. PMID:16809002

  8. Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan

    PubMed Central

    Scialò, Filippo; Sriram, Ashwin; Fernández-Ayala, Daniel; Gubina, Nina; Lõhmus, Madis; Nelson, Glyn; Logan, Angela; Cooper, Helen M.; Navas, Plácido; Enríquez, Jose Antonio; Murphy, Michael P.; Sanz, Alberto

    2016-01-01

    Summary Increased production of reactive oxygen species (ROS) has long been considered a cause of aging. However, recent studies have implicated ROS as essential secondary messengers. Here we show that the site of ROS production significantly contributes to their apparent dual nature. We report that ROS increase with age as mitochondrial function deteriorates. However, we also demonstrate that increasing ROS production specifically through respiratory complex I reverse electron transport extends Drosophila lifespan. Reverse electron transport rescued pathogenesis induced by severe oxidative stress, highlighting the importance of the site of ROS production in signaling. Furthermore, preventing ubiquinone reduction, through knockdown of PINK1, shortens lifespan and accelerates aging; phenotypes that are rescued by increasing reverse electron transport. These results illustrate that the source of a ROS signal is vital in determining its effects on cellular physiology and establish that manipulation of ubiquinone redox state is a valid strategy to delay aging. PMID:27076081

  9. Loss of c-Jun N-terminal kinase-interacting protein-1 does not affect axonal transport of the amyloid precursor protein or Aβ production

    PubMed Central

    Vagnoni, Alessio; Glennon, Elizabeth B.C.; Perkinton, Michael S.; Gray, Emma H.; Noble, Wendy; Miller, Christopher C.J.

    2013-01-01

    Disruption to axonal transport is an early pathological feature in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo in Alzheimer's disease since perturbation of its transport increases APP processing and production of amyloid-β peptide (Aβ) that is deposited in the brains of Alzheimer's disease patients. APP is transported anterogradely through axons on kinesin-1 motors. One favoured route for attachment of APP to kinesin-1 involves the scaffolding protein c-Jun N-terminal kinase-interacting protein-1 (JIP1), which has been shown to bind both APP and kinesin-1 light chain (KLC). However, direct experimental evidence to support a role of JIP1 in APP transport is lacking. Notably, the effect of loss of JIP1 on movement of APP through axons of living neurons, and the impact of such loss on APP processing and Aβ production has not been reported. To address these issues, we monitored how siRNA mediated loss of JIP1 influenced transport of enhanced green fluorescent protein (EGFP)-tagged APP through axons and production of endogenous Aβ in living neurons. Surprisingly, we found that knockdown of JIP1 did not affect either APP transport or Aβ production. These results have important implications for our understanding of APP trafficking in Alzheimer's disease. PMID:23825109

  10. 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

  11. HDAC6 Regulates Mitochondrial Transport in Hippocampal Neurons

    PubMed Central

    Chen, Sigeng; Owens, Geoffrey C.; Makarenkova, Helen; Edelman, David B.

    2010-01-01

    Background Tubulin is a major substrate of the cytoplasmic class II histone deacetylase HDAC6. Inhibition of HDAC6 results in higher levels of acetylated tubulin and enhanced binding of the motor protein kinesin-1 to tubulin, which promotes transport of cargoes along microtubules. Microtubule-dependent intracellular trafficking may therefore be regulated by modulating the activity of HDAC6. We have shown previously that the neuromodulator serotonin increases mitochondrial movement in hippocampal neurons via the Akt-GSK3β signaling pathway. Here, we demonstrate a role for HDAC6 in this signaling pathway. Methodology/Principal Findings We found that the presence of tubacin, a specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no effect. Compared to control cultures, higher levels of acetylated tubulin were found in neurons treated with tubacin, and more kinesin-1 was associated with mitochondria isolated from these neurons. Inhibition of GSK3β decreased cytoplasmic deacetylase activity and increased tubulin acetylation, whereas blockade of Akt, which phosphorylates and down-regulates GSK3β, increased cytoplasmic deacetylase activity and decreased tubulin acetylation. Concordantly, the administration of 5-HT, 8-OH-DPAT (a specific 5-HT1A receptor agonist), or fluoxetine (a 5-HT reuptake inhibitor) increased tubulin acetylation. GSK3β was found to co-localize with HDAC6 in hippocampal neurons, and inhibition of GSK3β resulted in decreased binding of antibody to phosphoserine-22, a potential GSK3β phosphorylation site in HDAC6. GSK3β may therefore regulate HDAC6 activity by phosphorylation. Conclusions/Significance This study demonstrates that HDAC6 plays an important role in the modulation of mitochondrial transport. The link between HDAC6 and GSK3β, established here, has important implications for our understanding of neurodegenerative disorders. In particular

  12. A HaloTag® method for assessing the retrograde axonal transport of the p75 neurotrophin receptor and other proteins in compartmented cultures of rat sympathetic neurons.

    PubMed

    Mok, Sue-Ann; Lund, Karen; Lapointe, Paul; Campenot, Robert B

    2013-03-30

    We have adapted HaloTag® (HT) technology for use in compartmented cultures of rat sympathetic neurons in order to provide a technique that can be broadly applied to studies of the retrograde transport of molecules that play roles in neurotrophin signaling. Transfected neurons expressing HT protein alone, HT protein fused to the p75 neurotrophin receptor (p75NTR) or HT protein fused to tubulin α-1B were maintained in compartmented cultures in which cell bodies and proximal axons of rat sympathetic neurons reside in proximal compartments and their distal axons extend into distal compartments. HT ligand containing a fluorescent tetramethylrhodamine (TMR) label was applied either in the distal compartments or the proximal compartments, and the transport of labeled proteins was assayed by gel fluorescence imaging and TMR immunoblot. HT protein expressed alone displayed little or no retrograde transport. HT protein fused to either the intracellular C-terminus or the extracellular N-terminus of p75NTR was retrogradely transported. The retrograde transport of p75NTR was augmented when the distal axons were provided with nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or antibodies to BDNF. The anterograde transport of HT protein fused to the N-terminus of tubulin α-1B was also demonstrated. We conclude that retrograde transport of HT fusion proteins provides a powerful and novel approach in studies of axonal transport. PMID:23348044

  13. The mitochondrial dicarboxylate and 2-oxoglutarate carriers do not transport glutathione

    PubMed Central

    Booty, Lee M.; King, Martin S.; Thangaratnarajah, Chancievan; Majd, Homa; James, Andrew M.; Kunji, Edmund R.S.; Murphy, Michael P.

    2015-01-01

    Glutathione carries out vital protective roles within mitochondria, but is synthesised in the cytosol. Previous studies have suggested that the mitochondrial dicarboxylate and 2-oxoglutarate carriers were responsible for glutathione uptake. We set out to characterise the putative glutathione transport by using fused membrane vesicles of Lactococcus lactis overexpressing the dicarboxylate and 2-oxoglutarate carriers. Although transport of the canonical substrates could be measured readily, an excess of glutathione did not compete for substrate uptake nor could transport of glutathione be measured directly. Thus these mitochondrial carriers do not transport glutathione and the identity of the mitochondrial glutathione transporter remains unknown. PMID:25637873

  14. The mitochondrial dicarboxylate and 2-oxoglutarate carriers do not transport glutathione.

    PubMed

    Booty, Lee M; King, Martin S; Thangaratnarajah, Chancievan; Majd, Homa; James, Andrew M; Kunji, Edmund R S; Murphy, Michael P

    2015-02-27

    Glutathione carries out vital protective roles within mitochondria, but is synthesised in the cytosol. Previous studies have suggested that the mitochondrial dicarboxylate and 2-oxoglutarate carriers were responsible for glutathione uptake. We set out to characterise the putative glutathione transport by using fused membrane vesicles of Lactococcus lactis overexpressing the dicarboxylate and 2-oxoglutarate carriers. Although transport of the canonical substrates could be measured readily, an excess of glutathione did not compete for substrate uptake nor could transport of glutathione be measured directly. Thus these mitochondrial carriers do not transport glutathione and the identity of the mitochondrial glutathione transporter remains unknown. PMID:25637873

  15. Overexpression of Mitochondrial Phosphate Transporter 3 Severely Hampers Plant Development through Regulating Mitochondrial Function in Arabidopsis

    PubMed Central

    Jia, Fengjuan; Wan, Xiaomin; Zhu, Wei; Sun, Dan; Zheng, Chengchao; Liu, Pei; Huang, Jinguang

    2015-01-01

    Mitochondria are abundant and important organelles present in nearly all eukaryotic cells, which maintain metabolic communication with the cytosol through mitochondrial carriers. The mitochondrial membrane localized phosphate transporter (MPT) plays vital roles in diverse development and signaling processes, especially the ATP biosynthesis. Among the three MPT genes in Arabidopsis genome, AtMPT3 was proven to be a major member, and its overexpression gave rise to multiple developmental defects including curly leaves with deep color, dwarfed stature, and reduced fertility. Transcript profiles revealed that genes involved in plant metabolism, cellular redox homeostasis, alternative respiration pathway, and leaf and flower development were obviously altered in AtMPT3 overexpression (OEMPT3) plants. Moreover, OEMPT3 plants also accumulated higher ATP content, faster respiration rate and more reactive oxygen species (ROS) than wild type plants. Overall, our studies showed that AtMPT3 was indispensable for Arabidopsis normal growth and development, and provided new sights to investigate its possible regulation mechanisms. PMID:26076137

  16. 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

  17. HSV, axonal transport and Alzheimer’s disease: in vitro and in vivo evidence for causal relationships

    PubMed Central

    Bearer, Elaine L

    2012-01-01

    HSV, a neurotropic virus, travels within neuronal processes by fast axonal transport. During neuronal infection HSV travels retrograde from the sensory nerve terminus to the neuronal cell body, where it replicates or enters latency. During replication HSV travels anterograde from the cell body to the nerve terminus. Postmortem studies find a high frequency of HSV DNA in the trigeminal ganglia as well as the brain. Studies correlating HSV with Alzheimer’s disease (AD) have been controversial. Here we review clinical evidence supporting such a link. Furthermore, the author describes experimental data showing physical interactions between nascent HSV particles and host transport machinery implicated in AD. The author concludes that the complexity of this relationship has been insufficiently explored, although the relative ease and nontoxicity of a potential anti-HSV treatment for AD demands further study. PMID:23335944

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

    PubMed

    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 Ins2(Akita) 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

  19. 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

  20. Chronic desipramine treatment alters tyrosine hydroxylase but not norepinephrine transporter immunoreactivity in norepinephrine axons in the rat prefrontal cortex

    PubMed Central

    Erickson, Susan L.; Gandhi, Anjalika R.; Asafu-Adjei, Josephine K.; Sampson, Allan R.; Miner, LeeAnn; Blakely, Randy D.; Sesack, Susan R.

    2011-01-01

    Pharmacological blockade of norepinephrine (NE) reuptake is clinically effective in treating several mental disorders. Drugs that bind to the NE transporter (NET) alter both protein levels and activity of NET and also the catecholamine synthetic enzyme tyrosine hydroxylase (TH). We examined the rat prefrontal cortex (PFC) by electron microscopy to determine whether the density and subcellular distribution of immunolabeling for NET and colocalization of NET with TH within individual NE axons were altered by chronic treatment with the selective NE uptake inhibitor desipramine (DMI). Following DMI treatment (21 days, 15 mg/kg/day), NET-immunoreactive (-ir) axons were significantly less likely to colocalize TH. This finding is consistent with reports of reduced TH levels and activity in the locus coeruleus after chronic DMI and indicates a reduction of NE synthetic capacity in the PFC. Measures of NET expression and membrane localization, including the number of NET-ir profiles per tissue area sampled, the number of gold particles per NET-ir profile area, and the proportion of gold particles associated with the plasma membrane, were similar in DMI and vehicle treated rats. These findings were verified using two different antibodies directed against distinct epitopes of the NET protein. The results suggest that chronic DMI treatment does not reduce NET expression within individual NE axons in vivo or induce an overall translocation of NET protein away from the plasma membrane in the PFC as measured by ultrastructural immunogold labeling. Our findings encourage consideration of possible postranslational mechanisms for regulating NET activity in antidepressant-induced modulation of NE clearance. PMID:21208501

  1. Axonal Targeting of the Serotonin Transporter in Cultured Rat Dorsal Raphe Neurons Is Specified by SEC24C-Dependent Export from the Endoplasmic Reticulum

    PubMed Central

    Sucic, Sonja; Koban, Florian; Schüchner, Stefan; Ogris, Egon; Sitte, Harald H.; Freissmuth, Michael

    2015-01-01

    Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT–607RI608–AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C–D796V/D797N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein–SERT from axons to the somatodendritic region. (4) SERT–K610Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes. PMID:24790205

  2. Axonal targeting of the serotonin transporter in cultured rat dorsal raphe neurons is specified by SEC24C-dependent export from the endoplasmic reticulum.

    PubMed

    Montgomery, Therese R; Steinkellner, Thomas; Sucic, Sonja; Koban, Florian; Schüchner, Stefan; Ogris, Egon; Sitte, Harald H; Freissmuth, Michael

    2014-04-30

    Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT-(607)RI(608)-AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C-D(796)V/D(797)N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein-SERT from axons to the somatodendritic region. (4) SERT-K(610)Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes. PMID:24790205

  3. Increased Expression of Reticulon 3 in Neurons Leads to Reduced Axonal Transport of β Site Amyloid Precursor Protein-cleaving Enzyme 1*

    PubMed Central

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

    2013-01-01

    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. PMID:24005676

  4. Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin

    PubMed Central

    Ashrafi, Ghazaleh; Schlehe, Julia S.; LaVoie, Matthew J.

    2014-01-01

    To minimize oxidative damage to the cell, malfunctioning mitochondria need to be removed by mitophagy. In neuronal axons, mitochondrial damage may occur in distal regions, far from the soma where most lysosomal degradation is thought to occur. In this paper, we report that PINK1 and Parkin, two Parkinson’s disease–associated proteins, mediate local mitophagy of dysfunctional mitochondria in neuronal axons. To reduce cytotoxicity and mimic physiological levels of mitochondrial damage, we selectively damaged a subset of mitochondria in hippocampal axons. Parkin was rapidly recruited to damaged mitochondria in axons followed by formation of LC3-positive autophagosomes and LAMP1-positive lysosomes. In PINK1−/− axons, damaged mitochondria did not accumulate Parkin and failed to be engulfed in autophagosomes. Similarly, initiation of mitophagy was blocked in Parkin−/− axons. Our findings demonstrate that the PINK1–Parkin-mediated pathway is required for local mitophagy in distal axons in response to focal damage. Local mitophagy likely provides rapid neuroprotection against oxidative stress without a requirement for retrograde transport to the soma. PMID:25154397

  5. Mitochondrial Electron Transport and Glycolysis are coupled in Articular Cartilage

    PubMed Central

    Martin, James A.; Martini, Anne; Molinari, Alexander; Morgan, Walter; Ramalingam, Wendy; Buckwalter, Joseph A.; McKinley, Todd O.

    2012-01-01

    Objective Although the majority of the ATP in chondrocytes is made by glycolysis rather than by oxidative phosphorylation in mitochondria there is evidence to suggest that reactive oxygen species produced by mitochondrial electron transport help to maintain cellular redox balance in favor of glycolysis. The objective of this study was to test this hypothesis by determining if rotenone, which inhibits electron transport and blocks oxidant production inhibits glycolytic ATP synthesis. Design Bovine osteochondral explants were treated with rotenone, an electron transport inhibitor; or oligomycin an ATP synthase inhibitor; or 2-fluoro-2-deoxy-D-glucose, a glycolysis inhibiter; or peroxide, an exogenous oxidant; or mitoquinone, a mitochondria-targeted anti-oxidant. Cartilage extracts were assayed for ATP, NAD+, and NADH, and culture medium was assayed for pyruvate and lactate after 24 hours of treatment. Imaging studies were used to measure superoxide production in cartilage. Results Rotenone and 2-fluoro-2-deoxy-D-glucose caused a significant decline in cartilage ATP (p < 0.001). In contrast, ATP levels were not affected by oligomycin. Peroxide treatment blocked rotenone effects on ATP, while treatment with MitoQ significantly suppressed ATP levels. Rotenone and 2-fluoro-2-deoxy-D-glucose caused a significant decline in pyruvate, but not in lactate production. NADH:NAD+ ratios decreased significantly in both rotenone and 2-fluoro-2-deoxy-D-glucose-treated explants (p < 0.05). Rotenone also significantly reduced superoxide production Conclusions These findings showing a link between glycolysis and electron transport are consistent with previous reports on the critical need for oxidants to support normal chondrocyte metabolism. They suggest a novel role for mitochondria in cartilage homeostasis that is independent of oxidative phosphorylation. PMID:22305999

  6. 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

  7. Functional expression and axonal transport of α7 nAChRs by peptidergic nociceptors of rat dorsal root ganglion.

    PubMed

    Shelukhina, Irina; Paddenberg, Renate; Kummer, Wolfgang; Tsetlin, Victor

    2015-07-01

    In recent pain studies on animal models, α7 nicotinic acetylcholine receptor (nAChR) agonists demonstrated analgesic, anti-hyperalgesic and anti-inflammatory effects, apparently acting through some peripheral receptors. Assuming possible involvement of α7 nAChRs on nociceptive sensory neurons, we investigated the morphological and neurochemical features of the α7 nAChR-expressing subpopulation of dorsal root ganglion (DRG) neurons and their ability to transport α7 nAChR axonally. In addition, α7 receptor activity and its putative role in pain signal neurotransmitter release were studied. Medium-sized α7 nAChR-expressing neurons prevailed, although the range covered all cell sizes. These cells accounted for one-fifth of total medium and large DRG neurons and <5% of small ones. 83.2% of α7 nAChR-expressing DRG neurons were peptidergic nociceptors (CGRP-immunopositive), one half of which had non-myelinated C-fibers and the other half had myelinated Aδ- and likely Aα/β-fibers, whereas 15.2% were non-peptidergic C-fiber nociceptors binding isolectin B4. All non-peptidergic and a third of peptidergic α7 nAChR-bearing nociceptors expressed TRPV1, a capsaicin-sensitive noxious stimulus transducer. Nerve crush experiments demonstrated that CGRPergic DRG nociceptors axonally transported α7 nAChRs both to the spinal cord and periphery. α7 nAChRs in DRG neurons were functional as their specific agonist PNU282987 evoked calcium rise enhanced by α7-selective positive allosteric modulator PNU120596. However, α7 nAChRs do not modulate neurotransmitter CGRP and glutamate release from DRG neurons since nicotinic ligands affected neither their basal nor provoked levels, showing the necessity of further studies to elucidate the true role of α7 nAChRs in those neurons. PMID:24706047

  8. Mitochondrial glutathione transport is a key determinant of neuronal susceptibility to oxidative and nitrosative stress.

    PubMed

    Wilkins, Heather M; Kirchhof, Danielle; Manning, Evan; Joseph, Jamie W; Linseman, Daniel A

    2013-02-15

    Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress. PMID:23283974

  9. Mitochondrial Glutathione Transport Is a Key Determinant of Neuronal Susceptibility to Oxidative and Nitrosative Stress*

    PubMed Central

    Wilkins, Heather M.; Kirchhof, Danielle; Manning, Evan; Joseph, Jamie W.; Linseman, Daniel A.

    2013-01-01

    Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress. PMID:23283974

  10. Mitochondrial Dysfunction in Amyotrophic Lateral Sclerosis

    PubMed Central

    Shi, Ping; Gal, Jozsef; Kwinter, David M.; Liu, Xiaoyan; Zhu, Haining

    2009-01-01

    The etiology of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains to be better understood. Based on the studies from ALS patients and transgenic animal models, it is believed that ALS is likely to be a multifactorial and multisystem disease. Many mechanisms have been postulated to be involved in the pathology of ALS, such as oxidative stress, glutamate excitotoxicity, mitochondrial damage, defective axonal transport, glia cell pathology and aberrant RNA metabolism. Mitochondria, which play crucial roles in excitotoxicity, apoptosis and cell survival, have shown to be an early target in ALS pathogenesis and contribute to the disease progression. Morphological and functional defects in mitochondria were found in both human patients and ALS mice overexpressing mutant SOD1. Mutant SOD1 was found to be preferentially associated with mitochondria and subsequently impair mitochondrial function. Recent studies suggest that axonal transport of mitochondria along microtubules and mitochondrial dynamics may also be disrupted in ALS. These results also illustrate the critical importance of maintaining proper mitochondrial function in axons and neuromuscular junctions, supporting the emerging “dying-back” axonopathy model of ALS. In this review, we will discuss how mitochondrial dysfunction has been linked to the ALS variants of SOD1 and the mechanisms by which mitochondrial damage contributes to the disease etiology. PMID:19715760

  11. Bioenergetic deficits in peripheral nerve sensory axons during chemotherapy-induced neuropathic pain resulting from peroxynitrite-mediated post-translational nitration of mitochondrial superoxide dismutase

    PubMed Central

    Janes, Kali; Doyle, Timothy; Bryant, Leesa; Esposito, Emanuela; Cuzzocrea, Salvatore; Ryerse, Jan; Bennett, Gary J.; Salvemini, Daniela

    2016-01-01

    Many of the widely used anticancer drugs induce dose-limiting peripheral neuropathies that undermine their therapeutic efficacy. Animal models of chemotherapy-induced painful peripheral neuropathy (CIPN) evoked by a variety of drug classes, including taxanes, vinca alkaloids, platinum-complexes, and proteasome-inhibitors, suggest that the common underlying mechanism in the development of these neuropathies is mitotoxicity in primary nerve sensory axons (PNSAs) arising from reduced mitochondrial bioenergetics [eg adenosine triphosphate (ATP) production deficits due to compromised respiratory complex I and II activity]. The causative mechanisms of this mitotoxicity remain poorly defined. However, peroxynitrite, an important pro-nociceptive agent, has been linked to mitotoxicity in several disease states and may also drive the mitotoxicity associated with CIPN. Our findings reveal that the development of mechano-hypersensitivity induced by paclitaxel, oxaliplatin, and bortezomib was prevented by administration of the peroxynitrite decomposition catalyst Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+) without interfering with their anti-tumor effects. Peak CIPN was associated with the nitration and inactivation of superoxide dismutase in the mitochondria, but not in the cytosol, as well as a significant decrease in ATP production within the PNSAs; all of these events were attenuated by MnTE-2-PyP5+. Our results provide continued support for the role of mitotoxicity in the development of CIPN across chemotherapeutic drug classes, and identify peroxynitrite as a key mediator in these processes, thereby providing the rationale towards development of “peroxynitrite-targeted” therapeutics for CIPN. PMID:23891899

  12. 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. PMID:14592946

  13. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.

    PubMed

    Mazarakis, N D; Azzouz, M; Rohll, J B; Ellard, F M; Wilkes, F J; Olsen, A L; Carter, E E; Barber, R D; Baban, D F; Kingsman, S M; Kingsman, A J; O'Malley, K; Mitrophanous, K A

    2001-09-15

    In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease. PMID:11590128

  14. Aluminum inhibits neurofilament assembly, cytoskeletal incorporation, and axonal transport. Dynamic nature of aluminum-induced perikaryal neurofilament accumulations as revealed by subunit turnover.

    PubMed

    Shea, T B; Wheeler, E; Jung, C

    1997-01-01

    The mechanism by which aluminum induces formation of perikaryal neurofilament (NF) inclusions remains unclear. Aluminum treatment inhibits: 1. The incorporation of newly synthesized NF subunits into Triton-insoluble cytoskeleton of axonal neurites; 2. Their degradation and dephosphorylation; 3. Their translocation into axonal neurites. It also fosters the accumulation of phosphorylated NFs within perikarya. In the present study, we addressed the relationship among these effects. Aluminum reduced the assembly of newly synthesized NF subunits into NFs. During examination of those subunits that did assemble in the presence of aluminum, it was revealed that aluminum also interfered with transport of newly assembled NFs into axonal neurites. Similarly, a delay in axonal transport of microinjected biotinylated NF-H was observed in aluminum-treated cells. Aluminum also inhibited the incorporation of newly synthesized and microinjected subunits into the Triton-insoluble cytoskeleton within both perikarya and neurites. Once incorporated into Triton-insoluble cytoskeletons, however, biotinylated subunits were retained within perikarya of aluminum-treated cells to a greater extent than within untreated cells. Notably, these subunits were depleted in the presence and absence of aluminum within 48 h, despite the persistence of the aluminum-induced perikaryal accumulation itself, suggesting that individual NF subunits undergo turnover even within aluminum-induced perikaryal accumulations. These findings demonstrate that aluminum interferes with multiple aspects of neurofilament dynamics and furthermore leaves open the possibility that aluminum-induced perikaryal NF whorls may not represent permanent structures, but rather may require continued recruitment of cytoskeletal constituents. PMID:9437656

  15. 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

  16. 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

  17. Mitochondrial transporters as novel targets for intracellular calcium signaling.

    PubMed

    Satrústegui, Jorgina; Pardo, Beatriz; Del Arco, Araceli

    2007-01-01

    Ca(2+) signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca(2+) entry in mitochondria via the Ca(2+) uniporter followed by Ca(2+) activation of three dehydrogenases in the mitochondrial matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca(2+) signals via a mechanism that does not require the entry of Ca(2+) in mitochondria, a mechanism depending on the activity of Ca(2+)-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC) and the ATP-Mg/P(i) carriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both types of CaMCs are activated by Ca(2+) in the intermembrane space and function together with the Ca(2+) uniporter in decoding the Ca(2+) signal into a mitochondrial response. PMID:17237342

  18. Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis

    PubMed Central

    Birk, A V; Chao, W M; Bracken, C; Warren, J D; Szeto, H H

    2014-01-01

    BACKGROUND AND PURPOSE Cardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c. EXPERIMENTAL APPROACH Interactions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria. KEY RESULTS SS-31 interacted only with liposomes and bicelles containing cardiolipin in about 1:1 ratio. NMR studies demonstrated that the aromatic residues of SS-31 penetrated deep into cardiolipin-containing bilayers. SS-31 restored cytochrome c reduction and mitochondrial oxygen consumption in the presence of added cardiolipin. In fresh mitochondria, SS-31 increased state 3 respiration and efficiency of ATP synthesis. CONCLUSIONS AND IMPLICATIONS SS-31 selectively targeted cardiolipin and modulated its interaction with cytochrome c. SS-31 inhibited the cytochrome c/cardiolipin complex peroxidase activity while protecting its ability to serve as an electron carrier, thus optimizing mitochondrial electron transport and ATP synthesis. This novel class of cardiolipin therapeutics has the potential to restore mitochondrial bioenergetics for treatment of numerous age-related diseases. LINKED ARTICLES This article is

  19. [Dysfunction of mitochondrial dynamic and distribution in Amyotrophic Lateral Sclerosis].

    PubMed

    Walczak, Jarosław; Szczepanowska, Joanna

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a complex disease leading to degradation of motor neurons. One of the early symptoms of many neurodegenerative disorders are mitochondrial dysfunctions. Since few decades mitochondrial morphology changes have been observed in tissues of patients with ALS. Mitochondria are highly dynamic organelles which constantly undergo continuous process of fusion and fission and are actively transported within the cell. Proper functioning of mitochondrial dynamics and distribution is crucial for cell survival, especially neuronal cells that have long axons. This article summarizes the current knowledge about the role of mitochondrial dynamics and distribution in pathophysiology of familial and sporadic form of ALS. PMID:26689011

  20. 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.

  1. Inner membrane fusion mediates spatial distribution of axonal mitochondria

    PubMed Central

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

    2016-01-01

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

  2. Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells.

    PubMed

    Granger, D L; Lehninger, A L

    1982-11-01

    Previous work has shown that injury of neoplastic cells by cytotoxic macrophages (CM) in cell culture is accompanied by inhibition of mitochondrial respiration. We have investigated the nature of this inhibition by studying mitochondrial respiration in CM-injured leukemia L1210 cells permeabilized with digitonin. CM-induced injury affects the mitochondrial respiratory chain proper. Complex I (NADH-coenzyme Q reductase) and complex II (succinate-coenzyme Q reductase) are markedly inhibited. In addition a minor inhibition of cytochrome oxidase was found. Electron transport from alpha-glycerophosphate through the respiratory chain to oxygen is unaffected and permeabilized CM-injured L1210 cells oxidizing this substrate exhibit acceptor control. However, glycerophosphate shuttle activity was found not to occur within CM-injured or uninjured L1210 cells in culture hence, alpha-glycerophosphate is apparently unavailable for mitochondrial oxidation in the intact cell. It is concluded that the failure of respiration of intact neoplastic cells injured by CM is caused by the nearly complete inhibition of complexes I and II of the mitochondrial electron transport chain. The time courses of CM-induced electron transport inhibition and arrest of L1210 cell division are examined and the possible relationship between these phenomena is discussed. PMID:6292238

  3. Glycoproteins gE and gI Are Required for Efficient KIF1A-Dependent Anterograde Axonal Transport of Alphaherpesvirus Particles in Neurons

    PubMed Central

    Kratchmarov, Radomir; Kramer, Tal; Greco, Todd M.; Taylor, Matthew P.; Ch'ng, Toh Hean; Cristea, Ileana M.

    2013-01-01

    Alphaherpesviruses, including pseudorabies virus (PRV), spread directionally within the nervous systems of their mammalian hosts. Three viral membrane proteins are required for efficient anterograde-directed spread of infection in neurons, including Us9 and a heterodimer composed of the glycoproteins gE and gI. We previously demonstrated that the kinesin-3 motor KIF1A mediates anterograde-directed transport of viral particles in axons of cultured peripheral nervous system (PNS) neurons. The PRV Us9 protein copurifies with KIF1A, recruiting the motor to transport vesicles, but at least one unidentified additional viral protein is necessary for this interaction. Here we show that gE/gI are required for efficient anterograde transport of viral particles in axons by mediating the interaction between Us9 and KIF1A. In the absence of gE/gI, viral particles containing green fluorescent protein (GFP)-tagged Us9 are assembled in the cell body but are not sorted efficiently into axons. Importantly, we found that gE/gI are necessary for efficient copurification of KIF1A with Us9, especially at early times after infection. We also constructed a PRV recombinant that expresses a functional gE-GFP fusion protein and used affinity purification coupled with mass spectrometry to identify gE-interacting proteins. Several viral and host proteins were found to associate with gE-GFP. Importantly, both gI and Us9, but not KIF1A, copurified with gE-GFP. We propose that gE/gI are required for efficient KIF1A-mediated anterograde transport of viral particles because they indirectly facilitate or stabilize the interaction between Us9 and KIF1A. PMID:23804637

  4. Review: Axon pathology in age-related neurodegenerative disorders.

    PubMed

    Adalbert, R; Coleman, M P

    2013-02-01

    'Dying back' axon degeneration is a prominent feature of many age-related neurodegenerative disorders and is widespread in normal ageing. Although the mechanisms of disease- and age-related losses may differ, both contribute to symptoms. Here, we review recent advances in understanding axon pathology in age-related neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and glaucoma. In particular, we highlight the importance of axonal transport, autophagy, traumatic brain injury and mitochondrial quality control. We then place these disease mechanisms in the context of changes to axons and dendrites that occur during normal ageing. We discuss what makes ageing such an important risk factor for many neurodegenerative disorders and conclude that the processes of normal ageing and disease combine at the molecular, cellular or systems levels in a range of disorders to produce symptoms. Pathology identical to disease also occurs at the cellular level in most elderly individuals. Thus, normal ageing and age-related disease are inextricably linked and the term 'healthy ageing' downplays the important contributions of cellular pathology. For a full understanding of normal ageing or age-related disease we must study both processes. PMID:23046254

  5. Comparison of Retinal Nerve Fiber Layer Thickness In Vivo and Axonal Transport after Chronic Intraocular Pressure Elevation in Young versus Older Rats

    PubMed Central

    Abbott, Carla J.; Choe, Tiffany E.; Burgoyne, Claude F.; Cull, Grant; Wang, Lin; Fortune, Brad

    2014-01-01

    Purpose To compare in young and old rats longitudinal measurements of retinal nerve fiber layer thickness (RNFLT) and axonal transport 3-weeks after chronic IOP elevation. Method IOP was elevated unilaterally in 2- and 9.5-month-old Brown-Norway rats by intracameral injections of magnetic microbeads. RNFLT was measured by spectral domain optical coherence tomography. Anterograde axonal transport was assessed from confocal scanning laser ophthalmolscopy of superior colliculi (SC) after bilateral intravitreal injections of cholera toxin-B-488. Optic nerve sections were graded for damage. Results Mean IOP was elevated in both groups (young 37, old 38 mmHg, p = 0.95). RNFL in young rats exhibited 10% thickening at 1-week (50.9±8.1 µm, p<0.05) vs. baseline (46.4±2.4 µm), then 7% thinning at 2-weeks (43.0±7.2 µm, p>0.05) and 3-weeks (43.5±4.4 µm, p>0.05), representing 20% loss of dynamic range. RNFLT in old rats showed no significant change at 1-week (44.9±4.1 µm) vs. baseline (49.2±5.3 µm), but progression to 22% thinning at 2-weeks (38.0±3.7 µm, p<0.01) and 3-weeks (40.0±6.6 µm, p<0.05), representing 59% loss of dynamic range. Relative SC fluorescence intensity was reduced in both groups (p<0.001), representing 77–80% loss of dynamic range and a severe transport deficit. Optic nerves showed 75–95% damage (p<0.001). There was greater RNFL thinning in old rats (p<0.05), despite equivalent IOP insult, transport deficit and nerve damage between age groups (all p>0.05). Conclusion Chronic IOP elevation resulted in severely disrupted axonal transport and optic nerve axon damage in all rats, associated with mild RNFL loss in young rats but a moderate RNFL loss in old rats despite the similar IOP insult. Hence, the glaucomatous injury response within the RNFL depends on age. PMID:25501362

  6. IMPORTANCE OF MITOCHONDRIAL PO2 IN MAXIMAL O2 TRANSPORT AND UTILIZATION: A THEORETICAL ANALYSIS

    PubMed Central

    Cano, I; Mickael, M; Gomez-Cabrero, D.; Tegnér, J; Roca, J; Wagner, PD

    2013-01-01

    In previous calculations of how the O2 transport system limits V̇O2max, it was reasonably assumed that mitochondrial PO2 (PmO2) could be neglected (set to zero). However, in reality, PmO2 must exceed zero and the red cell to mitochondrion diffusion gradient may therefore be reduced, impairing diffusive transport of O2 and V̇O2max. Accordingly, we investigated the influence of PmO2 on these calculations by coupling previously used equations for O2 transport to one for mitochondrial respiration relating mitochondrial V̇O2 to PO2. This hyperbolic function, characterized by its P50 and V̇MAX, allowed PmO2 to become a model output (rather than set to zero as previously). Simulations using data from exercising normal subjects showed that at V̇O2max, PmO2was usually < 1 mm Hg, and that the effects on V̇O2max were minimal. However, when O2 transport capacity exceeded mitochondrial V̇MAX, or if P50 were elevated, PmO2 often reached double digit values, thereby reducing the diffusion gradient and significantly decreasing V̇O2max. PMID:24012990

  7. 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

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

    PubMed

    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

  9. 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

    Zhang, Jingyi; McDonald, Alexander J

    2016-04-01

    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

  10. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence

    PubMed Central

    Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana; Jensen, Jennifer; Michailowsky, Vladimir; Andrews, Norma W.

    2016-01-01

    Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes. PMID:26741360

  11. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

    PubMed

    Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana; Jensen, Jennifer; Michailowsky, Vladimir; Andrews, Norma W

    2016-01-01

    Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes. PMID:26741360

  12. Subcellular Localization Determines the Stability and Axon Protective Capacity of Axon Survival Factor Nmnat2

    PubMed Central

    Milde, Stefan; Gilley, Jonathan; Coleman, Michael P.

    2013-01-01

    Axons require a constant supply of the labile axon survival factor Nmnat2 from their cell bodies to avoid spontaneous axon degeneration. Here we investigate the mechanism of fast axonal transport of Nmnat2 and its site of action for axon maintenance. Using dual-colour live-cell imaging of axonal transport in SCG primary culture neurons, we find that Nmnat2 is bidirectionally trafficked in axons together with markers of the trans-Golgi network and synaptic vesicles. In contrast, there is little co-migration with mitochondria, lysosomes, and active zone precursor vesicles. Residues encoded by the small, centrally located exon 6 are necessary and sufficient for stable membrane association and vesicular axonal transport of Nmnat2. Within this sequence, a double cysteine palmitoylation motif shared with GAP43 and surrounding basic residues are all required for efficient palmitoylation and stable association with axonal transport vesicles. Interestingly, however, disrupting this membrane association increases the ability of axonally localized Nmnat2 to preserve transected neurites in primary culture, while re-targeting the strongly protective cytosolic mutants back to membranes abolishes this increase. Larger deletions within the central domain including exon 6 further enhance Nmnat2 axon protective capacity to levels that exceed that of the slow Wallerian degeneration protein, WldS. The mechanism underlying the increase in axon protection appears to involve an increased half-life of the cytosolic forms, suggesting a role for palmitoylation and membrane attachment in Nmnat2 turnover. We conclude that Nmnat2 activity supports axon survival through a site of action distinct from Nmnat2 transport vesicles and that protein stability, a key determinant of axon protection, is enhanced by mutations that disrupt palmitoylation and dissociate Nmnat2 from these vesicles. PMID:23610559

  13. Why do axons differ in caliber?

    PubMed Central

    Perge, János A.; Niven, Jeremy E.; Mugnaini, Enrico; Balasubramanian, Vijay; Sterling, Peter

    2012-01-01

    CNS axons differ in diameter (d) by nearly 100-fold (~ 0.1 to 10μm); therefore they differ in cross-sectional area (d2) and volume by nearly 10,000-fold. If, as found for optic nerve, mitochondrial volume-fraction is constant with axon diameter, energy capacity would rise with axon volume, also as d2. Given constraints on space and energy, we asked what functional requirements set an axon’s diameter? Surveying 16 fiber groups spanning nearly the full range of diameters in five species (guinea pig, rat, monkey, locust, octopus), we found that: (i) thin axons are most numerous; (ii) mean firing frequencies, estimated for 9 of the identified axon classes, are low for thin fibers and high for thick ones, ranging from ~1 to >100Hz; (iii) a tract’s distribution of fiber diameters, whether narrow or broad, and whether symmetric or skewed, reflects heterogeneity of information rates conveyed by its individual fibers; (iv) mitochondrial volume/axon length, rises ≥ d2. To explain the pressure towards thin diameters we note an established law of diminishing returns: an axon, to double its information rate, must more than double its firing rate. Since diameter is apparently linear with firing rate, doubling information rate would more than quadruple an axon’s volume and energy use. Thicker axons may be needed to encode features that cannot be efficiently decoded if their information is spread over several low-rate channels. Thus information rate may be the main variable that sets axon caliber - with axons constrained to deliver information at the lowest acceptable rate. PMID:22238098

  14. Giant Axonal Neuropathy

    MedlinePlus

    ... Diversity Find People About NINDS NINDS Giant Axonal Neuropathy Information Page Table of Contents (click to jump ... done? Clinical Trials Organizations What is Giant Axonal Neuropathy? Giant axonal neuropathy (GAN) is a rare inherited ...

  15. 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.

  16. Caloric restriction improves efficiency and capacity of the mitochondrial electron transport chain in Saccharomyces cerevisiae

    SciTech Connect

    Choi, Joon-Seok; Choi, Kyung-Mi; Lee, Cheol-Koo

    2011-06-03

    Highlights: {yields} Calorie restriction (CR) increases electron transport chain (ETC) at both RNA and protein level. {yields} CR enhances mitochondrial membrane potential, and, regardless of ages, reduces reactive oxygen species. {yields} CR increases both efficiency and capacity of the ETC. {yields} CR induces intensive modulation at mitochondrial ETC where might be a major site leading to extension of lifespan. -- Abstract: Caloric restriction (CR) is known to extend lifespan in a variety of species; however, the mechanism remains unclear. In this study, we found that CR potentiated the mitochondrial electron transport chain (ETC) at both the transcriptional and translational levels. Indeed, mitochondrial membrane potential (MMP) was increased by CR, and, regardless of ages, overall reactive oxygen species (ROS) generation was decreased by CR. With these changes, overall growth rate of cells was maintained under various CR conditions, just like cells under a non-restricted condition. All of these data support increased efficiency and capacity of the ETC by CR, and this change might lead to extension of lifespan.

  17. Characterizing MttA as a mitochondrial cis-aconitic acid transporter by metabolic engineering.

    PubMed

    Steiger, Matthias G; Punt, Peter J; Ram, Arthur F J; Mattanovich, Diethard; Sauer, Michael

    2016-05-01

    The mitochondrial carrier protein MttA is involved in the biosynthesis of itaconic acid in Aspergillus terreus. In this paper, the transport specificity of MttA is analyzed making use of different metabolically engineered Aspergillus niger strains. Furthermore, the mitochondrial localization of this protein is confirmed using fluorescence microscopy. It was found that MttA preferentially transports cis-aconitic acid over citric acid and does not transport itaconic acid. The expression of MttA in selected A. niger strains results in secretion of aconitic acid. MttA can be used in further strain engineering strategies to transport cis-aconitic acid to the cytosol to produce itaconic acid or related metabolites. The microbial production of aconitic acid (9g/L) is achieved in strains expressing this transport protein. Thus, metabolic engineering can be used for both the in vivo characterization of transport protein function like MttA and to make use of this protein by creating aconitic acid producing strains. PMID:26875555

  18. Gem1 and ERMES Do Not Directly Affect Phosphatidylserine Transport from ER to Mitochondria or Mitochondrial Inheritance

    PubMed Central

    Nguyen, Tammy T; Lewandowska, Agnieszka; Choi, Jae-Yeon; Markgraf, Daniel F; Junker, Mirco; Bilgin, Mesut; Ejsing, Christer S; Voelker, Dennis R; Rapoport, Tom A; Shaw, Janet M

    2012-01-01

    In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology. PMID:22409400

  19. A biophysical analysis of mitochondrial movement: differences between transport in neuronal cell bodies versus processes

    PubMed Central

    Narayanareddy, Babu Reddy Janakaloti; Vartiainen, Suvi; Hariri, Neema; O’Dowd, Diane K.; Gross, Steven P.

    2014-01-01

    There is increasing interest in factors that can impede cargo transport by molecular motors inside the cell. While potentially relevant (1), the importance of cargo size and sub-cellular location have received relatively little attention. Here we address these questions taking advantage of the fact that mitochondria—a common cargo—in Drosophila neurons exhibit a wide distribution of sizes. In addition, the mitochondria can be genetically marked with GFP making it possible to visualize and compare their movement in the cell bodies and processes of living cells. Using total internal reflection (TIRF) microscopy coupled with particle tracking and analysis, we quantified transport properties of GFP positive mitochondria as a function of their size and location. In neuronal cell bodies we find little evidence for significant opposition to motion, consistent with a previous study on lipid droplets (2). However, in the processes we observe an inverse relationship between mitochondrial size and velocity and run distances. This can be ameliorated via hypotonic treatment to increase process size, suggesting that motor mediated movement is impeded in this more confined environment. Interestingly, we also observe local mitochondrial accumulations in processes but not in cell bodies. Such accumulations do not completely block transport, but do increase the probability of mitochondria-mitochondria interactions. They are thus particularly interesting in relation to mitochondrial exchange of elements. PMID:24673933

  20. 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. PMID:26398944

  1. 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

  2. Mitochondrial traffic jams in Alzheimer's disease - pinpointing the roadblocks.

    PubMed

    Correia, Sónia C; Perry, George; Moreira, Paula I

    2016-10-01

    The vigorous axonal transport of mitochondria, which serves to distribute these organelles in a dynamic and non-uniform fashion, is crucial to fulfill neuronal energetic requirements allowing the maintenance of neurons structure and function. Particularly, axonal transport of mitochondria and their spatial distribution among the synapses are directly correlated with synaptic activity and integrity. Despite the basis of Alzheimer's disease (AD) remains enigmatic, axonal pathology and synaptic dysfunction occur prior the occurrence of amyloid-β (Aβ) deposition and tau aggregation, the two classical hallmarks of this devastating neurodegenerative disease. Importantly, the early stages of AD are marked by defects on axonal transport of mitochondria as denoted by the abnormal accumulation of mitochondria within large swellings along dystrophic and degenerating neuritis. Within this scenario, this review is devoted to identify the molecular "roadblocks" underlying the abnormal axonal transport of mitochondria and consequent synaptic "starvation" and neuronal degeneration in AD. Understanding the molecular nature of defective mitochondrial transport may provide a new avenue to counteract AD pathology. PMID:27460705

  3. Mitochondrial Efficiency-Dependent Viability of Saccharomyces cerevisiae Mutants Carrying Individual Electron Transport Chain Component Deletions.

    PubMed

    Kwon, Young-Yon; Choi, Kyung-Mi; Cho, ChangYeon; Lee, Cheol-Koo

    2015-12-31

    Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 (sdh1Δ, sdh2Δ, sdh4Δ, cor1Δ, cyt1Δ, qcr7Δ, qcr8Δ, rip1Δ, cox6Δ, cox7Δ, cox9Δ, atp4Δ, atp7Δ, and atp17Δ) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-F1F0-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency. PMID:26608359

  4. Homodimeric Intrinsic Membrane Proteins. Identification and Modulation of Interactions between Mitochondrial Transporter (Carrier) Subunits

    PubMed Central

    Wohlrab, Hartmut

    2010-01-01

    Transporter (carrier) proteins of the inner mitochondrial membrane link metabolic pathways within the matrix and the cytosol with transport/exchange of metabolites and inorganic ions. Their strict control of these fluxes is required for oxidative phosphorylation. Understanding the ternary complex transport mechanism with which most of these transporters function requires an accounting of the number and interactions of their subunits. The phosphate transporter (PTP, Mir1p) subunit readily forms homodimers with intersubunit affinities changeable by mutations. Cys28, likely at the subunit interface, is a site for mutations yielding transport inhibition or a channel-like transport mode. Such mutations yield a small increase or decrease in affinity between the subunits. The PTP inhibitor N-ethylmaleimide decreases subunit affinity by a small amount. PTP mutations that yield the highest (40%) and the lowest (2%) liposome incorporation efficiencies (LIE) are clustered near Cys28. Such mutant subunits show the lowest and highest subunit affinities respectively. The oxaloacetate transporter (Oac1p) subunit has an almost 2-fold lower affinity than the PTP subunit. The Oac1p, dicarboxylate (Dic1p) and PTP transporter subunits form heterodimers with even lower affinities. These results form a firm basis for detailed studies to establish the effect of subunit affinities on transport mode and activity and for the identification of the mechanism that prevents formation of heterodimers that surely will negatively impact oxidative phosphorylation and ATP levels with serious consequences for the cell. PMID:20171189

  5. Neuronal influences are necessary to produce mitochondrial co-localization with glutamate transporters in astrocytes

    PubMed Central

    Ugbode, Christopher I; Hirst, Warren D; Rattray, Marcus

    2014-01-01

    Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial:GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments.Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity. PMID

  6. Human Genetic Disorders of Axon Guidance

    PubMed Central

    Engle, Elizabeth C.

    2010-01-01

    This article reviews symptoms and signs of aberrant axon connectivity in humans, and summarizes major human genetic disorders that result, or have been proposed to result, from defective axon guidance. These include corpus callosum agenesis, L1 syndrome, Joubert syndrome and related disorders, horizontal gaze palsy with progressive scoliosis, Kallmann syndrome, albinism, congenital fibrosis of the extraocular muscles type 1, Duane retraction syndrome, and pontine tegmental cap dysplasia. Genes mutated in these disorders can encode axon growth cone ligands and receptors, downstream signaling molecules, and axon transport motors, as well as proteins without currently recognized roles in axon guidance. Advances in neuroimaging and genetic techniques have the potential to rapidly expand this field, and it is feasible that axon guidance disorders will soon be recognized as a new and significant category of human neurodevelopmental disorders. PMID:20300212

  7. 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

  8. Unique function of Kinesin Kif5A in localization of mitochondria in axons.

    PubMed

    Campbell, Philip D; Shen, Kimberle; Sapio, Matthew R; Glenn, Thomas D; Talbot, William S; Marlow, Florence L

    2014-10-29

    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

  9. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration

    PubMed Central

    Tang-Schomer, Min D.; Patel, Ankur R.; Baas, Peter W.; Smith, Douglas H.

    2010-01-01

    Little is known about which components of the axonal cytoskeleton might break during rapid mechanical deformation, such as occurs in traumatic brain injury. Here, we micropatterned neuronal cell cultures on silicone membranes to induce dynamic stretch exclusively of axon fascicles. After stretch, undulating distortions formed along the axons that gradually relaxed back to a straight orientation, demonstrating a delayed elastic response. Subsequently, swellings developed, leading to degeneration of almost all axons by 24 h. Stabilizing the microtubules with taxol maintained the undulating geometry after injury but greatly reduced axon degeneration. Conversely, destabilizing microtubules with nocodazole prevented undulations but greatly increased the rate of axon loss. Ultrastructural analyses of axons postinjury revealed immediate breakage and buckling of microtubules in axon undulations and progressive loss of microtubules. Collectively, these data suggest that dynamic stretch of axons induces direct mechanical failure at specific points along microtubules. This microtubule disorganization impedes normal relaxation of the axons, resulting in undulations. However, this physical damage also triggers progressive disassembly of the microtubules around the breakage points. While the disintegration of microtubules allows delayed recovery of the “normal” straight axon morphology, it comes at a great cost by interrupting axonal transport, leading to axonal swelling and degeneration.—Tang-Schomer, M. D., Patel, A. R,, Baas, P. W., Smith, D. H. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration. PMID:20019243

  10. Mitochondrial Citrate Transporter-dependent Metabolic Signature in the 22q11.2 Deletion Syndrome.

    PubMed

    Napoli, Eleonora; Tassone, Flora; Wong, Sarah; Angkustsiri, Kathleen; Simon, Tony J; Song, Gyu; Giulivi, Cecilia

    2015-09-18

    The congenital disorder 22q11.2 deletion syndrome (22qDS), characterized by a hemizygous deletion of 1.5-3 Mb on chromosome 22 at locus 11.2, is the most common microdeletion disorder (estimated prevalence of 1 in 4000) and the second risk factor for schizophrenia. Nine of ∼30 genes involved in 22qDS have the potential of disrupting mitochondrial metabolism (COMT, UFD1L, DGCR8, MRPL40, PRODH, SLC25A1, TXNRD2, T10, and ZDHHC8). Deficits in bioenergetics during early postnatal brain development could set the basis for a disrupted neuronal metabolism or synaptic signaling, partly explaining the higher incidence in developmental and behavioral deficits in these individuals. Here, we investigated whether mitochondrial outcomes and metabolites from 22qDS children segregated with the altered dosage of one or several of these mitochondrial genes contributing to 22qDS etiology and/or morbidity. Plasma metabolomics, lymphocytic mitochondrial outcomes, and epigenetics (histone H3 Lys-4 trimethylation and 5-methylcytosine) were evaluated in samples from 11 22qDS children and 13 age- and sex-matched neurotypically developing controls. Metabolite differences between 22qDS children and controls reflected a shift from oxidative phosphorylation to glycolysis (higher lactate/pyruvate ratios) accompanied by an increase in reductive carboxylation of α-ketoglutarate (increased concentrations of 2-hydroxyglutaric acid, cholesterol, and fatty acids). Altered metabolism in 22qDS reflected a critical role for the haploinsufficiency of the mitochondrial citrate transporter SLC25A1, further enhanced by HIF-1α, MYC, and metabolite controls. This comprehensive profiling served to clarify the biochemistry of this disease underlying its broad, complex phenotype. PMID:26221035

  11. Consequences of defective vitamin A transportation on mitochondrial membrane integrity during protein depletion.

    PubMed

    Olowookere, J O

    1986-01-01

    The relationships between the structural integrity and functionality of rat liver mitochondrial membranes, and different levels of dietary protein and vitamin A transportation during protein depletion in animals have been investigated. Although the vitamin A content of the protein-depleted diet was 1680 +/- 35 IU/kg diet, and that of the control diet was 1,650 +/- 30 IU/kg diet, the vitamin A content of the liver of depleted rats was reduced to 16.7% of controls. The hepatic mitochondria of rats fed a protein-depleted diet showed excessive passive swelling (about 3-fold of controls) in isotonic solutions. Whereas a seemingly inverse relationship existed between the vitamin A content of the liver and the osmotic behaviour of hepatic mitochondria of rats fed a protein-depleted diet, there is a direct relationship between their hepatic mitochondrial vitamin A and the respiratory control ratio. The implications of these observations are discussed. PMID:3717896

  12. Heinrich Wieland--prize lecture. Transport of proteins across mitochondrial membranes.

    PubMed

    Neupert, W

    1994-03-01

    mitochondrial processing peptidase which cleaves signal sequences after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle. PMID:8043971

  13. Tumor cell death induced by the inhibition of mitochondrial electron transport: The effect of 3-hydroxybakuchiol

    SciTech Connect

    Jaña, Fabián; Faini, Francesca; Lapier, Michel; Pavani, Mario; Kemmerling, Ulrike; Morello, Antonio; Maya, Juan Diego; Jara, José; Parra, Eduardo; Ferreira, Jorge

    2013-10-15

    Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells. - Highlights: • We studied the anticancer activity of a natural compound, 3-OHbk, on TA3/Ha cells. • 3-OHbk inhibited mitochondrial electron flow by interacting with Complex I. • Complex I inhibition did

  14. Reduced number of axonal mitochondria and tau hypophosphorylation in mouse P301L tau knockin neurons

    PubMed Central

    Rodríguez-Martín, Teresa; Pooler, Amy M.; Lau, Dawn H.W.; Mórotz, Gábor M.; De Vos, Kurt J.; Gilley, Jonathan; Coleman, Michael P.; Hanger, Diane P.

    2016-01-01

    Expression of the frontotemporal dementia-related tau mutation, P301L, at physiological levels in adult mouse brain (KI-P301L mice) results in overt hypophosphorylation of tau and age-dependent alterations in axonal mitochondrial transport in peripheral nerves. To determine the effects of P301L tau expression in the central nervous system, we examined the kinetics of mitochondrial axonal transport and tau phosphorylation in primary cortical neurons from P301L knock-in (KI-P301L) mice. We observed a significant 50% reduction in the number of mitochondria in the axons of cortical neurons cultured from KI-P301L mice compared to wild-type neurons. Expression of murine P301L tau did not change the speed, direction of travel or likelihood of movement of mitochondria. Notably, the angle that defines the orientation of the mitochondria in the axon, and the volume of individual moving mitochondria, were significantly increased in neurons expressing P301L tau. We found that murine tau phosphorylation in KI-P301L mouse neurons was diminished and the ability of P301L tau to bind to microtubules was also reduced compared to tau in wild-type neurons. The P301L mutation did not influence the ability of murine tau to associate with membranes in cortical neurons or in adult mouse brain. We conclude that P301L tau is associated with mitochondrial changes and causes an early reduction in murine tau phosphorylation in neurons coupled with impaired microtubule binding of tau. These results support the association of mutant tau with detrimental effects on mitochondria and will be of significance for the pathogenesis of tauopathies. PMID:26459111

  15. Neurofilament spacing, phosphorylation, and axon diameter in regenerating and uninjured lamprey axons.

    PubMed

    Pijak, D S; Hall, G F; Tenicki, P J; Boulos, A S; Lurie, D I; Selzer, M E

    1996-05-13

    uncut GRAs but were also highly phosphorylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF sidearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, such as the slowing of NF transport and the formation of a stationary cytoskeletal lattice, as has been proposed by others. Such a mechanism could be overridden during regeneration, when a more compact, phosphorylated NF backbone might add mechanical stiffness that promotes the advance of the neurite tip within a restricted central nervous system environment. PMID:8744444

  16. The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids.

    PubMed

    Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

    2014-05-01

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation. PMID:24652292

  17. Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

    PubMed Central

    Tomac, A; Widenfalk, J; Lin, L F; Kohno, T; Ebendal, T; Hoffer, B J; Olson, L

    1995-01-01

    The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons. Images Fig. 2 Fig. 3 Fig. 4 PMID:7667281

  18. Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

    PubMed Central

    Nguyen, Tammy T.; Oh, Sang S.; Weaver, David; Lewandowska, Agnieszka; Maxfield, Dane; Schuler, Max-Hinderk; Smith, Nathan K.; Macfarlane, Jane; Saunders, Gerald; Palmer, Cheryl A.; Debattisti, Valentina; Koshiba, Takumi; Pulst, Stefan; Feldman, Eva L.; Hajnóczky, György; Shaw, Janet M.

    2014-01-01

    Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease. PMID:25136135

  19. Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease.

    PubMed

    Nguyen, Tammy T; Oh, Sang S; Weaver, David; Lewandowska, Agnieszka; Maxfield, Dane; Schuler, Max-Hinderk; Smith, Nathan K; Macfarlane, Jane; Saunders, Gerald; Palmer, Cheryl A; Debattisti, Valentina; Koshiba, Takumi; Pulst, Stefan; Feldman, Eva L; Hajnóczky, György; Shaw, Janet M

    2014-09-01

    Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease. PMID:25136135

  20. Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.

    PubMed

    Titov, Denis V; Cracan, Valentin; Goodman, Russell P; Peng, Jun; Grabarek, Zenon; Mootha, Vamsi K

    2016-04-01

    A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state. PMID:27124460

  1. Pseudophosphorylation of tau at S422 enhances SDS-stable dimer formation and impairs both anterograde and retrograde fast axonal transport.

    PubMed

    Tiernan, Chelsea T; Combs, Benjamin; Cox, Kristine; Morfini, Gerardo; Brady, Scott T; Counts, Scott E; Kanaan, Nicholas M

    2016-09-01

    In Alzheimer's disease (AD), tau undergoes numerous modifications, including increased phosphorylation at serine-422 (pS422). In the human brain, pS422 tau protein is found in prodromal AD, correlates well with cognitive decline and neuropil thread pathology, and appears associated with increased oligomer formation and exposure of the N-terminal phosphatase-activating domain (PAD). However, whether S422 phosphorylation contributes to toxic mechanisms associated with disease-related forms of tau remains unknown. Here, we report that S422-pseudophosphorylated tau (S422E) lengthens the nucleation phase of aggregation without altering the extent of aggregation or the types of aggregates formed. When compared to unmodified tau aggregates, the S422E modification significantly increased the amount of SDS-stable tau dimers, despite similar levels of immunoreactivity with an oligomer-selective antibody (TOC1) and another antibody that reports PAD exposure (TNT1). Vesicle motility assays in isolated squid axoplasm further revealed that S422E tau monomers inhibited anterograde, kinesin-1 dependent fast axonal transport (FAT). Unexpectedly, and unlike unmodified tau aggregates, which selectively inhibit anterograde FAT, aggregates composed of S422E tau were found to inhibit both anterograde and retrograde FAT. Highlighting the relevance of these findings to human disease, pS422 tau was found to colocalize with tau oligomers and with a fraction of tau showing increased PAD exposure in the human AD brain. This study identifies novel effects of pS422 on tau biochemical properties, including prolonged nucleation and enhanced dimer formation, which correlate with a distinct inhibitory effect on FAT. Taken together, these findings identify a novel mechanistic basis by which pS422 confers upon tau a toxic effect that may directly contribute to axonal dysfunction in AD and other tauopathies. PMID:27373205

  2. Mitochondrial Mislocalization Underlies Aβ42-Induced Neuronal Dysfunction in a Drosophila Model of Alzheimer's Disease

    PubMed Central

    Iijima-Ando, Kanae; Hearn, Stephen A.; Shenton, Christopher; Gatt, Anthony; Zhao, LiJuan; Iijima, Koichi

    2009-01-01

    The amyloid-β 42 (Aβ42) is thought to play a central role in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanisms by which Aβ42 induces neuronal dysfunction and degeneration remain elusive. Mitochondrial dysfunctions are implicated in AD brains. Whether mitochondrial dysfunctions are merely a consequence of AD pathology, or are early seminal events in AD pathogenesis remains to be determined. Here, we show that Aβ42 induces mitochondrial mislocalization, which contributes to Aβ42-induced neuronal dysfunction in a transgenic Drosophila model. In the Aβ42 fly brain, mitochondria were reduced in axons and dendrites, and accumulated in the somata without severe mitochondrial damage or neurodegeneration. In contrast, organization of microtubule or global axonal transport was not significantly altered at this stage. Aβ42-induced behavioral defects were exacerbated by genetic reductions in mitochondrial transport, and were modulated by cAMP levels and PKA activity. Levels of putative PKA substrate phosphoproteins were reduced in the Aβ42 fly brains. Importantly, perturbations in mitochondrial transport in neurons were sufficient to disrupt PKA signaling and induce late-onset behavioral deficits, suggesting a mechanism whereby mitochondrial mislocalization contributes to Aβ42-induced neuronal dysfunction. These results demonstrate that mislocalization of mitochondria underlies the pathogenic effects of Aβ42 in vivo. PMID:20016833

  3. Mitochondrial Electron Transport Chain in Heavy Metal-Induced Neurotoxicity: Effects of Cadmium, Mercury, and Copper

    PubMed Central

    Belyaeva, Elena A.; Sokolova, Tatyana V.; Emelyanova, Larisa V.; Zakharova, Irina O.

    2012-01-01

    To clarify the role of mitochondrial electron transport chain (mtETC) in heavy-metal-induced neurotoxicity, we studied action of Cd2+, Hg2+, and Cu2+ on cell viability, intracellular reactive oxygen species formation, respiratory function, and mitochondrial membrane potential of rat cell line PC12. As found, the metals produced, although in a different way, dose- and time-dependent changes of all these parameters. Importantly, Cd2+ beginning from 10 [mu]M and already at short incubation time (3 h) significantly inhibited the FCCP-uncoupled cell respiration; besides, practically the complete inhibition of the respiration was reached after 3 h incubation with 50 [mu]M Hg2+ or 500 [mu]M Cd2+, whereas even after 48 h exposure with 500 [mu]M Cu2+, only a 50% inhibition of the respiration occurred. Against the Cd2+-induced cell injury, not only different antioxidants and mitochondrial permeability transition pore inhibitors were protective but also such mtETC effectors as FCCP and stigmatellin (complex III inhibitor). However, all mtETC effectors used did not protect against the Hg2+- or Cu2+-induced cell damage. Notably, stigmatellin was shown to be one of the strongest protectors against the Cd2+-induced cell damage, producing a 15–20% increase in the cell viability. The mechanisms of the mtETC involvement in the heavy-metal-induced mitochondrial membrane permeabilization and cell death are discussed. PMID:22619586

  4. Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes.

    PubMed

    Di Noia, Maria Antonietta; Van Driesche, Sarah; Palmieri, Ferdinando; Yang, Li-Ming; Quan, Shuo; Goodman, Alvin I; Abraham, Nader G

    2006-06-01

    Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity. PMID:16595661

  5. Increased production of mitochondrial superoxide in the spinal cord induces pain behaviors in mice: the effect of mitochondrial electron transport complex inhibitors.

    PubMed

    Kim, Hee Young; Chung, Jin Mo; Chung, Kyungsoon

    2008-12-01

    Scavengers of reactive oxygen species (ROS) have been shown to produce a strong antinociceptive effect on persistent pain, and mitochondria are suggested to be the main source of ROS in the spinal dorsal horn. To explore whether excessive generation of mitochondrial superoxide alone can induce pain, the effect of mitochondrial electron transport complex inhibitors on the development of mechanical hyperalgesia was examined in mice. Intrathecal injection of an electron transport complex inhibitor, antimycin A or rotenone, in normal mice resulted in a slowly developing but long-lasting and dose-dependent mechanical hyperalgesia. The levels of mechanical hyperalgesia after antimycin A, a complex III inhibitor, were higher than that with rotenone, a complex I inhibitor. A large increase of mitochondrial superoxide in the spinal dorsal horn and a strong antinociceptive effect of ROS scavengers, phenyl-N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were observed in antimycin A-treated mice. The study indicates that the enhanced production of spinal mitochondrial superoxide alone without nerve injury can produce mechanical hyperalgesia. PMID:18832013

  6. Mitochondrial Myopathies

    MedlinePlus

    ... line and are therefore called the electron transport chain, and complex V actually churns out ATP, so ... coQ10 , is a component of the electron transport chain, which uses oxygen to manufacture ATP. Some mitochondrial ...

  7. Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements.

    PubMed

    Reynafarje, B; Brand, M D; Lehninger, A L

    1976-12-10

    The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0. PMID:12164

  8. Amyloid β oligomers elicit mitochondrial transport defects and fragmentation in a time-dependent and pathway-specific manner.

    PubMed

    Rui, Yanfang; Zheng, James Q

    2016-01-01

    Small oligomeric forms of amyloid-β (Aβ) are believed to be the culprit for declined brain functions in AD in part through their impairment of neuronal trafficking and synaptic functions. However, the precise cellular actions of Aβ oligomers and underlying mechanisms in neurons remain to be fully defined. Previous studies have identified mitochondria as a major target of Aβ toxicity contributing to early cognitive decline and memory loss in neurodegenerative diseases including Alzheimer's disease (AD). In this study, we report that Aβ oligomers acutely elicit distinct effects on the transport and integrity of mitochondria. We found that acute exposure of hippocampal neurons to Aβ oligomers from either synthetic peptides or AD brain homogenates selectively impaired fast transport of mitochondria without affecting the movement of late endosomes and lysosomes. Extended exposure of hipoocampal neurons to Aβ oligomers was found to result in mitochondrial fragmentation. While both mitochondrial effects induced by Aβ oligomers can be abolished by the inhibition of GSK3β, they appear to be independent from each other. Aβ oligomers impaired mitochondrial transport through HDAC6 activation whereas the fragmentation involved the GTPase Drp-1. These results show that Aβ oligomers can acutely disrupt mitochondrial transport and integrity in a time-dependent and pathway-specific manner. These findings thus provide new insights into Aβ-induced mitochondrial defects that may contribute to neuronal dysfunction and AD pathogenesis. PMID:27535553

  9. Mitochondrial fission is an acute and adaptive response in injured motor neurons.

    PubMed

    Kiryu-Seo, Sumiko; Tamada, Hiromi; Kato, Yukina; Yasuda, Katsura; Ishihara, Naotada; Nomura, Masatoshi; Mihara, Katsuyoshi; Kiyama, Hiroshi

    2016-01-01

    Successful recovery from neuronal damage requires a huge energy supply, which is provided by mitochondria. However, the physiological relevance of mitochondrial dynamics in damaged neurons in vivo is poorly understood. To address this issue, we established unique bacterial artificial chromosome transgenic (BAC Tg) mice, which develop and function normally, but in which neuronal injury induces labelling of mitochondria with green fluorescent protein (GFP) and expression of cre recombinase. GFP-labelled mitochondria in BAC Tg mice appear shorter in regenerating motor axons soon after nerve injury compared with mitochondria in non-injured axons, suggesting the importance of increased mitochondrial fission during the early phase of nerve regeneration. Crossing the BAC Tg mice with mice carrying a floxed dynamin-related protein 1 gene (Drp1), which is necessary for mitochondrial fission, ablates mitochondrial fission specifically in injured neurons. Injury-induced Drp1-deficient motor neurons show elongated or abnormally gigantic mitochondria, which have impaired membrane potential and axonal transport velocity during the early phase after injury, and eventually promote neuronal death. Our in vivo data suggest that acute and prominent mitochondrial fission during the early stage after nerve injury is an adaptive response and is involved in the maintenance of mitochondrial and neuronal integrity to prevent neurodegeneration. PMID:27319806

  10. Mitochondrial fission is an acute and adaptive response in injured motor neurons

    PubMed Central

    Kiryu-Seo, Sumiko; Tamada, Hiromi; Kato, Yukina; Yasuda, Katsura; Ishihara, Naotada; Nomura, Masatoshi; Mihara, Katsuyoshi; Kiyama, Hiroshi

    2016-01-01

    Successful recovery from neuronal damage requires a huge energy supply, which is provided by mitochondria. However, the physiological relevance of mitochondrial dynamics in damaged neurons in vivo is poorly understood. To address this issue, we established unique bacterial artificial chromosome transgenic (BAC Tg) mice, which develop and function normally, but in which neuronal injury induces labelling of mitochondria with green fluorescent protein (GFP) and expression of cre recombinase. GFP-labelled mitochondria in BAC Tg mice appear shorter in regenerating motor axons soon after nerve injury compared with mitochondria in non-injured axons, suggesting the importance of increased mitochondrial fission during the early phase of nerve regeneration. Crossing the BAC Tg mice with mice carrying a floxed dynamin-related protein 1 gene (Drp1), which is necessary for mitochondrial fission, ablates mitochondrial fission specifically in injured neurons. Injury-induced Drp1-deficient motor neurons show elongated or abnormally gigantic mitochondria, which have impaired membrane potential and axonal transport velocity during the early phase after injury, and eventually promote neuronal death. Our in vivo data suggest that acute and prominent mitochondrial fission during the early stage after nerve injury is an adaptive response and is involved in the maintenance of mitochondrial and neuronal integrity to prevent neurodegeneration. PMID:27319806

  11. DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites.

    PubMed

    Norkett, Rosalind; Modi, Souvik; Birsa, Nicol; Atkin, Talia A; Ivankovic, Davor; Pathania, Manav; Trossbach, Svenja V; Korth, Carsten; Hirst, Warren D; Kittler, Josef T

    2016-01-01

    The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder, and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but its effects in dendrites have yet to be studied. Further, the mechanisms of this regulation and its role in neuronal development and brain function are poorly understood. Here we have demonstrated that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane GTPase proteins Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins (mitofusins). Using live cell imaging, we show that disruption of the DISC1-Miro-TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localizes to the mitochondria, where it similarly disrupts mitochondrial dynamics. We also show by super resolution microscopy that DISC1 is localized to endoplasmic reticulum contact sites and that the DISC1-Boymaw fusion protein decreases the endoplasmic reticulum-mitochondria contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro-TRAK complex or upon expression of the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator of mitochondrial dynamics in both axons and dendrites to mediate the transport, fusion, and cross-talk of these organelles, and pathological DISC1 isoforms disrupt this critical function leading to abnormal neuronal development. PMID:26553875

  12. DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites*

    PubMed Central

    Norkett, Rosalind; Modi, Souvik; Birsa, Nicol; Atkin, Talia A.; Ivankovic, Davor; Pathania, Manav; Trossbach, Svenja V.; Korth, Carsten; Hirst, Warren D.; Kittler, Josef T.

    2016-01-01

    The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder, and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but its effects in dendrites have yet to be studied. Further, the mechanisms of this regulation and its role in neuronal development and brain function are poorly understood. Here we have demonstrated that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane GTPase proteins Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins (mitofusins). Using live cell imaging, we show that disruption of the DISC1-Miro-TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localizes to the mitochondria, where it similarly disrupts mitochondrial dynamics. We also show by super resolution microscopy that DISC1 is localized to endoplasmic reticulum contact sites and that the DISC1-Boymaw fusion protein decreases the endoplasmic reticulum-mitochondria contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro-TRAK complex or upon expression of the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator of mitochondrial dynamics in both axons and dendrites to mediate the transport, fusion, and cross-talk of these organelles, and pathological DISC1 isoforms disrupt this critical function leading to abnormal neuronal development. PMID:26553875

  13. Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution.

    PubMed Central

    Agrimi, G; Di Noia, M A; Marobbio, C M T; Fiermonte, G; Lasorsa, F M; Palmieri, F

    2004-01-01

    The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene. PMID:14674884

  14. 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

  15. Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

    PubMed Central

    Eya, Jonathan C.; Ukwuaba, Vitalis O.; Yossa, Rodrigue; Gannam, Ann L.

    2015-01-01

    A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish. PMID:25853266

  16. Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants.

    PubMed

    Vigani, Gianpiero; Bashir, Khurram; Ishimaru, Yasuhiro; Lehmann, Martin; Casiraghi, Fabio Marco; Nakanishi, Hiromi; Seki, Motoaki; Geigenberger, Peter; Zocchi, Graziano; Nishizawa, Naoko K

    2016-03-01

    Iron (Fe) is an essential micronutrient for plant growth and development, and its reduced bioavailability strongly impairs mitochondrial functionality. In this work, the metabolic adjustment in the rice (Oryza sativa) mitochondrial Fe transporter knockdown mutant (mit-2) was analysed. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared with wild-type (WT) plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases accumulated strongly in mit-2 plants, indicating that alternative pathways were activated to keep the respiratory chain working. Additionally, large-scale changes in the transcriptome and metabolome were observed in mit-2 rice plants. In particular, a strong alteration (up-/down-regulation) in the expression of genes encoding enzymes of both primary and secondary metabolism was found in mutant plants. This was reflected by changes in the metabolic profiles in both roots and shoots of mit-2 plants. Significant alterations in the levels of amino acids belonging to the aspartic acid-related pathways (aspartic acid, lysine, and threonine in roots, and aspartic acid and ornithine in shoots) were found that are strictly connected to the Krebs cycle. Furthermore, some metabolites (e.g. pyruvic acid, fumaric acid, ornithine, and oligosaccharides of the raffinose family) accumulated only in the shoot of mit-2 plants, indicating possible hypoxic responses. These findings suggest that the induction of local Fe deficiency in the mitochondrial compartment of mit-2 plants differentially affects the transcript as well as the metabolic profiles in root and shoot tissues. PMID:26685186

  17. Biphasic modulation of the mitochondrial electron transport chain in myocardial ischemia and reperfusion

    PubMed Central

    Lee, Hsin-Ling; Chen, Chwen-Lih; Yeh, Steve T.; Zweier, Jay L.

    2012-01-01

    Mitochondrial electron transport chain (ETC) is the major source of reactive oxygen species during myocardial ischemia-reperfusion (I/R) injury. Ischemic defect and reperfusion-induced injury to ETC are critical in the disease pathogenesis of postischemic heart. The properties of ETC were investigated in an isolated heart model of global I/R. Rat hearts were subjected to ischemia for 30 min followed by reperfusion for 1 h. Studies of mitochondrial function indicated a biphasic modulation of electron transfer activity (ETA) and ETC protein expression during I/R. Analysis of ETAs in the isolated mitochondria indicated that complexes I, II, III, and IV activities were diminished after 30 min of ischemia but increased upon restoration of flow. Immunoblotting analysis and ultrastructural analysis with transmission electron microscopy further revealed marked downregulation of ETC in the ischemic heart and then upregulation of ETC upon reperfusion. No significant difference in the mRNA expression level of ETC was detected between ischemic and postischemic hearts. However, reperfusion-induced ETC biosynthesis in myocardium can be inhibited by cycloheximide, indicating the involvement of translational control. Immunoblotting analysis of tissue homogenates revealed a similar profile in peroxisome proliferator-activated receptor-γ coactivator-1α expression, suggesting its essential role as an upstream regulator in controlling ETC biosynthesis during I/R. Significant impairment caused by ischemic and postischemic injury was observed in the complexes I- III. Analysis of NADH ferricyanide reductase activity indicated that injury of flavoprotein subcomplex accounts for 50% decline of intact complex I activity from ischemic heart. Taken together, our findings provide a new insight into the molecular mechanism of I/R-induced mitochondrial dysfunction. PMID:22268109

  18. Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants

    PubMed Central

    Vigani, Gianpiero; Bashir, Khurram; Ishimaru, Yasuhiro; Lehmann, Martin; Casiraghi, Fabio Marco; Nakanishi, Hiromi; Seki, Motoaki; Geigenberger, Peter; Zocchi, Graziano; Nishizawa, Naoko K.

    2016-01-01

    Iron (Fe) is an essential micronutrient for plant growth and development, and its reduced bioavailability strongly impairs mitochondrial functionality. In this work, the metabolic adjustment in the rice (Oryza sativa) mitochondrial Fe transporter knockdown mutant (mit-2) was analysed. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared with wild-type (WT) plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases accumulated strongly in mit-2 plants, indicating that alternative pathways were activated to keep the respiratory chain working. Additionally, large-scale changes in the transcriptome and metabolome were observed in mit-2 rice plants. In particular, a strong alteration (up-/down-regulation) in the expression of genes encoding enzymes of both primary and secondary metabolism was found in mutant plants. This was reflected by changes in the metabolic profiles in both roots and shoots of mit-2 plants. Significant alterations in the levels of amino acids belonging to the aspartic acid-related pathways (aspartic acid, lysine, and threonine in roots, and aspartic acid and ornithine in shoots) were found that are strictly connected to the Krebs cycle. Furthermore, some metabolites (e.g. pyruvic acid, fumaric acid, ornithine, and oligosaccharides of the raffinose family) accumulated only in the shoot of mit-2 plants, indicating possible hypoxic responses. These findings suggest that the induction of local Fe deficiency in the mitochondrial compartment of mit-2 plants differentially affects the transcript as well as the metabolic profiles in root and shoot tissues. PMID:26685186

  19. Stable over-expression of the 2-oxoglutarate carrier enhances neuronal cell resistance to oxidative stress via Bcl-2-dependent mitochondrial GSH transport

    PubMed Central

    Wilkins, Heather M.; Brock, Samantha; Gray, Josie J.; Linseman, Daniel A.

    2015-01-01

    Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron-like cell lines over-expressing the mitochondrial GSH transporter, the 2-oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up-regulation of the anti-apoptotic protein, B cell lymphoma 2 (Bcl-2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl-2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co-immunoprecipitated with Bcl-2 from rat brain lysates in a GSH-dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl-2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels. PMID:24606213

  20. Stable over-expression of the 2-oxoglutarate carrier enhances neuronal cell resistance to oxidative stress via Bcl-2-dependent mitochondrial GSH transport.

    PubMed

    Wilkins, Heather M; Brock, Samantha; Gray, Josie J; Linseman, Daniel A

    2014-07-01

    Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron-like cell lines over-expressing the mitochondrial GSH transporter, the 2-oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up-regulation of the anti-apoptotic protein, B cell lymphoma 2 (Bcl-2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl-2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co-immunoprecipitated with Bcl-2 from rat brain lysates in a GSH-dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl-2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels. Stable over-expression of the 2-oxoglutarate carrier (OGC) in a motor neuronal cell line induced a specific increase in mitochondrial GSH and markedly enhanced resistance to oxidative stress. Over-expression of OGC also induced Bcl-2 expression which was owing to the specific increase in mitochondrial GSH. Intriguingly, enhanced expression of Bcl-2 was required to sustain OGC-dependent GSH transport into the mitochondria. Thus, OGC and Bcl-2 work in a concerted manner to maintain the

  1. Aspirin, acetaminophen and proton transport through phospholipid bilayers and mitochondrial membranes.

    PubMed

    Gutknecht, J

    1992-09-01

    Mechanisms of proton transport were investigated in planar phospholipid bilayer membranes exposed to aspirin (acetylsalicylic acid), acetaminophen (4-acetamidophenol), benzoic acid and three aspirin metabolites (salicylic acid, gentisic acid and salicyluric acid). The objectives were to characterize the conductances and permeabilities of these weak acids in lipid bilayer membranes and then predict their effects on mitochondrial membranes. Of the compounds tested only aspirin, benzoate and salicylate caused significant increases in membrane conductance. The conductance was due mainly to proton current at low pH and to weak acid anion current at neutral pH. Analysis of the concentration and pH dependence suggests that these weak acids act as HA-2-type proton carriers when pH approximately pK and as lipid soluble anions at neutral pH. Salicylate is much more potent than aspirin and benzoate because salicylate contains an internal hydrogen bond which delocalizes the negative charge and increases the permeability of the anion. Model calculations for mitochondria suggest that salicylate causes net H+ uptake by a cyclic process of HA influx and A- efflux. This model can explain the salicylate-induced uncoupling and swelling observed in isolated mitochondria. Since ingested aspirin breaks down rapidly to form salicylate, these results may clarify the mechanisms of aspirin toxicity in humans. The results may also help to explain why the ingestion of aspirin but not acetaminophen is associated with Reye's syndrome, a disease characterized by impaired energy metabolism and mitochondrial swelling. PMID:1334228

  2. The transport machinery for the import of preproteins across the outer mitochondrial membrane.

    PubMed

    Ryan, M T; Wagner, R; Pfanner, N

    2000-01-01

    In order for proteins to be imported into subcellular compartments, they must first traverse the organellar membranes. In mitochondria, hydrophilic protein channels in both the outer and inner membranes serve such a purpose. Recently, the channel protein of the outer mitochondrial membrane was identified to be Tom40. Tom40 is found in a high molecular weight complex termed the general import pore (GIP) complex where it is tightly associated with the receptor protein Tom22 along with Tom7, Tom6 and Tom5. Tom7 and Tom6 seem to modulate the dynamics of the GIP complex while Tom5 is involved in preprotein transfer from receptors to Tom40. The receptor proteins Tom70 and Tom20 associate with this complex in a weaker manner where they are involved in the initial recognition of preproteins. This review focuses on the identification and characterisation of the transport machinery of the outer mitochondrial membrane and how they are involved in the co-ordination and regulation of events required for the translocation of preproteins into mitochondria. PMID:10661891

  3. Structural transition in Bcl-xL and its potential association with mitochondrial calcium ion transport

    PubMed Central

    Rajan, Sreekanth; Choi, Minjoo; Nguyen, Quoc Toan; Ye, Hong; Liu, Wei; Toh, Hui Ting; Kang, CongBao; Kamariah, Neelagandan; Li, Chi; Huang, Huiya; White, Carl; Baek, Kwanghee; Grüber, Gerhard; Yoon, Ho Sup

    2015-01-01

    Bcl-2 family proteins are key regulators for cellular homeostasis in response to apoptotic stimuli. Bcl-xL, an antiapoptotic Bcl-2 family member, undergoes conformational transitions, which leads to two conformational states: the cytoplasmic and membrane-bound. Here we present the crystal and small-angle X-ray scattering (SAXS) structures of Bcl-xL treated with the mild detergent n-Octyl β-D-Maltoside (OM). The detergent-treated Bcl-xL forms a dimer through three-dimensional domain swapping (3DDS) by swapping helices α6-α8 between two monomers. Unlike Bax, a proapoptotic member of the Bcl-2 family, Bcl-xL is not converted to 3DDS homodimer upon binding BH3 peptides and ABT-737, a BH3 mimetic drug. We also designed Bcl-xL mutants which cannot dimerize and show that these mutants reduced mitochondrial calcium uptake in MEF cells. This illustrates the structural plasticity in Bcl-xL providing hints toward the probable molecular mechanism for Bcl-xL to play a regulatory role in mitochondrial calcium ion transport. PMID:26023881

  4. Accumulation of [3H]fucose-labelled glycoproteins in the Golgi apparatus of dorsal root ganglion neurons during inhibition of fast axonal transport caused by exposure of the ganglion to Co2+-containing or Ca2+-free medium.

    PubMed

    Lavoie, P A; Bennett, G

    1983-01-01

    Previous in vitro studies have established that Co2+-containing or Ca2+-free media interfere with the initiation of the fast axonal transport of proteins. The present study has used light- and electron-microscope radioautography to compare the distribution of [3H]fucose-labelled glycoproteins in neuronal cell bodies of control dorsal root ganglia and ganglia incubated for 16-17 h in Ca2+-free medium or in medium containing 0.18 mM Co2+. The radioautographic reaction in control cell bodies was diffusely scattered throughout the cytoplasm; grain counts revealed that 22% of the reaction was associated with elements of the Golgi apparatus and 78% was over other organelles and the remainder of the cytoplasm. In most experimental cell bodies, 78% of the silver grains were clustered over elements of the Golgi complex whereas other organelles and the remainder of the cytoplasm were comparatively much less labelled; structural alterations of the Golgi apparatus were also produced by the modified media. In parallel studies where the radioactivity in nerve trunks and ganglia was measured by liquid scintillation counting, it was found that the Ca2+-free medium and the Co2+-containing medium both reduced by approximately 80% the quantity of [3H]fucose-labelled glycoproteins which were carried by the fast axonal transport system; they did so without interfering with the incorporation of [3H]fucose into glycoproteins. The results indicate that in the presence of Co2+ or in the absence of Ca2+ the proteins which are destined for fast axonal transport accumulate at the Golgi apparatus of neuronal cell bodies. These results thus suggest that Ca2+ is required for proteins to leave the Golgi region in transit to the fast axonal transport system. PMID:6188994

  5. 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.

  6. Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation.

    PubMed

    Emelyanova, Larisa; Ashary, Zain; Cosic, Milanka; Negmadjanov, Ulugbek; Ross, Gracious; Rizvi, Farhan; Olet, Susan; Kress, David; Sra, Jasbir; Tajik, A Jamil; Holmuhamedov, Ekhson L; Shi, Yang; Jahangir, Arshad

    2016-07-01

    Mitochondria are critical for maintaining normal cardiac function, and a deficit in mitochondrial energetics can lead to the development of the substrate that promotes atrial fibrillation (AF) and its progression. However, the link between mitochondrial dysfunction and AF in humans is still not fully defined. The aim of this study was to elucidate differences in the functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes and oxidative stress in right atrial tissue from patients without (non-AF) and with AF (AF) who were undergoing open-heart surgery and were not significantly different for age, sex, major comorbidities, and medications. The overall functional activity of the electron transport chain (ETC), NADH:O2 oxidoreductase activity, was reduced by 30% in atrial tissue from AF compared with non-AF patients. This was predominantly due to a selective reduction in complex I (0.06 ± 0.007 vs. 0.09 ± 0.006 nmol·min(-1)·citrate synthase activity(-1), P = 0.02) and II (0.11 ± 0.012 vs. 0.16 ± 0.012 nmol·min(-1)·citrate synthase activity(-1), P = 0.003) functional activity in AF patients. Conversely, complex V activity was significantly increased in AF patients (0.21 ± 0.027 vs. 0.12 ± 0.01 nmol·min(-1)·citrate synthase activity(-1), P = 0.005). In addition, AF patients exhibited a higher oxidative stress with increased production of mitochondrial superoxide (73 ± 17 vs. 11 ± 2 arbitrary units, P = 0.03) and 4-hydroxynonenal level (77.64 ± 30.2 vs. 9.83 ± 2.83 ng·mg(-1) protein, P = 0.048). Our findings suggest that AF is associated with selective downregulation of ETC activity and increased oxidative stress that can contribute to the progression of the substrate for AF. PMID:27199126

  7. Vesicular Glutamate (VGluT), GABA (VGAT), and Acetylcholine (VAChT) Transporters in Basal Forebrain Axon Terminals Innervating the Lateral Hypothalamus

    PubMed Central

    HENNY, PABLO; JONES, BARBARA E.

    2008-01-01

    The basal forebrain (BF) is known to play important roles in cortical activation and sleep, which are likely mediated by chemically differentiated cell groups including cholinergic, γ-aminobutyric acid (GABA)ergic and other unidentified neurons. One important target of these cells is the lateral hypothalamus (LH), which is critical for arousal and the maintenance of wakefulness. To determine whether chemically specific BF neurons provide an innervation to the LH, we employed anterograde transport of 10,000 MW biotinylated dextran amine (BDA) together with immunohistochemical staining of the vesicular transporter proteins (VTPs) for glutamate (VGluT1, -2, and -3), GABA (VGAT), or acetylcholine (ACh, VAChT). In addition, we applied triple staining for the postsynaptic proteins (PSPs), PSD-95 with VGluT or Gephyrin (Geph) with VGAT, to examine whether the BDA-labeled varicosities may form excitatory or inhibitory synapses in the LH. Axons originating from BDA-labeled neurons in the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) descended within the medial forebrain bundle and extended collateral varicose fibers to contact LH neurons. In the LH, the BDA-labeled varicosities were immunopositive (+) for VAChT (~10%), VGluT2 (~25%), or VGAT (~50%), revealing an important influence of newly identified glutamatergic together with GABAergic BF inputs. Moreover, in confocal microscopy, VGluT2+ and VGAT+ terminals were apposed to PSD-95+ and Geph+ profiles respectively, indicating that they formed synaptic contacts with LH neurons. The important inputs from glutamatergic and GABAergic BF cells could thus regulate LH neurons in an opposing manner to stimulate vs. suppress cortical activation and behavioral arousal reciprocally. PMID:16572456

  8. Development of a mitochondrial respiratory electron transport bioindicator for assessment of aromatic hydrocarbon toxicity

    SciTech Connect

    Tripuranthakam, S.; Duxbury, C.L.; Babu, T.S.; Greenberg, B.M.

    1999-07-01

    Polycyclic aromatic hydrocarbons (PAHs) are phototoxic to animals and plants. To monitor the toxicity of PAHs and understand their mechanism(s) of action, it is important to develop rapid and accurate bioindicators of effect. In this study, the authors have analyzed the impact of the anthracene photooxidation product, 1,2-dihydroxyanthraquinone (1,2-dhATQ), on mitochondrial electron transport using a preparation of beef heart mitochondria. Employing both NADH and succinate as electron donors, it was found that 1,2-dhATQ inhibited respiratory electron transport to cytochrome c. Using reduced DCPIP (2,6 dichlorophenolindophenol) as an electron donor, it was found that electron transfer was also inhibited. This indicated that inhibition was specific to cytochrome bc{sub 1} (ubiquinone-cytochrome c oxidoreductase). Inhibition of cytochrome c oxidase was very weak, indicating that 1,2-dhATQ primarily targets the cytochrome bc{sub 1} complex. Thus, analysis of the respiratory electron transport revealed a specific site of impact for 1,2-dhATQ, and a potential mechanistic basis for toxicity of this compound.

  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. Reductions in kinesin expression are associated with nitric oxide-induced axonal damage.

    PubMed

    Redondo, Juliana; Hares, Kelly; Wilkins, Alastair; Scolding, Neil; Kemp, Kevin

    2015-06-01

    Axonal injury is often characterized by axonal transport defects and abnormal accumulation of intra-axonal components. Nitric oxide (NO) has a key role in mediating inflammatory axonopathy in many neurodegenerative diseases, but little is known about how nitrosative/oxidative stress affects axonal transport or whether reductions in kinesin superfamily protein (KIF) expression correlate with axon pathology. KIFs are molecular motors that have a key role in axonal and dendritic transport, and impairment of these mechanisms has been associated with a number of neurological disorders. This study shows that rat cortical neurons exposed to NO display both a time-dependent decrease in KIF gene/protein expression and neurofilament phosphorylation in addition to a reduction in axonal length and neuronal survival. Because mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for neuronal/axonal repair, this study analyzes the capacity of MSCs to protect neurons and axonal transport mechanisms from NO damage. Results show that coculture of MSCs with NO-exposed neurons results in the preservation of KIF expression, axonal length, and neuronal survival. Altogether, these results suggest a potential mechanism involved in the disruption of axonal transport and abnormal accumulation of proteins in axons during nitrosative insult. We hypothesize that impaired axonal transport contributes, per se, to progression of injury and provide further evidence of the therapeutic potential of MSCs for neurodegenerative disorders. PMID:25639260