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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. 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

  3. Mitochondrial fission augments capsaicin-induced axonal degeneration.

    PubMed

    Chiang, Hao; Ohno, Nobuhiko; Hsieh, Yu-Lin; Mahad, Don J; Kikuchi, Shin; Komuro, Hitoshi; Hsieh, Sung-Tsang; Trapp, Bruce D

    2015-01-01

    Capsaicin, an agonist of transient receptor potential vanilloid receptor 1, induces axonal degeneration of peripheral sensory nerves and is commonly used to treat painful sensory neuropathies. In this study, we investigated the role of mitochondrial dynamics in capsaicin-induced axonal degeneration. In capsaicin-treated rodent sensory axons, axonal swellings, decreased mitochondrial stationary site length and reduced mitochondrial transport preceded axonal degeneration. Increased axoplasmic Ca(2+) mediated the alterations in mitochondrial length and transport. While sustaining mitochondrial transport did not reduce axonal swellings in capsaicin-treated axons, preventing mitochondrial fission by overexpression of mutant dynamin-related protein 1 increased mitochondrial length, retained mitochondrial membrane potentials and reduced axonal loss upon capsaicin treatment. These results establish that mitochondrial stationary site size significantly affects axonal integrity and suggest that inhibition of Ca(2+)-dependent mitochondrial fission facilitates mitochondrial function and axonal survival following activation of axonal cationic channels.

  4. The axonal transport of mitochondria

    PubMed Central

    Saxton, William M.; Hollenbeck, Peter J.

    2012-01-01

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

  5. Miro, MCU, and calcium: bridging our understanding of mitochondrial movement in axons.

    PubMed

    Niescier, Robert F; Chang, Karen T; Min, Kyung-Tai

    2013-09-10

    Neurons are extremely polarized structures with long axons and dendrites, which require proper distribution of mitochondria and maintenance of mitochondrial dynamics for neuronal functions and survival. Indeed, recent studies show that various neurological disorders are linked to mitochondrial transport in neurons. Mitochondrial anterograde transport is believed to deliver metabolic energy to synaptic terminals where energy demands are high, while mitochondrial retrograde transport is required to repair or remove damaged mitochondria in axons. It has been suggested that Ca(2) (+) plays a key role in regulating mitochondrial transport by altering the configuration of mitochondrial protein, miro. However, molecular mechanisms that regulate mitochondrial transport in neurons still are not well characterized. In this review, we will discuss the roles of miro in mitochondrial transport and how the recently identified components of the mitochondrial calcium uniporter add to our current model of mitochondrial mobility regulation.

  6. 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

  7. 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

  8. 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

  9. 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

  10. A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons.

    PubMed

    Aschrafi, Armaz; Kar, Amar N; Gale, Jenna R; Elkahloun, Abdel G; Vargas, Jose Noberto S; Sales, Naomi; Wilson, Gabriel; Tompkins, Miranda; Gioio, Anthony E; Kaplan, Barry B

    2016-09-01

    Mitochondria are enriched in subcellular regions of high energy consumption, such as axons and pre-synaptic nerve endings. Accumulating evidence suggests that mitochondrial maintenance in these distal structural/functional domains of the neuron depends on the "in-situ" translation of nuclear-encoded mitochondrial mRNAs. In support of this notion, we recently provided evidence for the axonal targeting of several nuclear-encoded mRNAs, such as cytochrome c oxidase, subunit 4 (COXIV) and ATP synthase, H+ transporting and mitochondrial Fo complex, subunit C1 (ATP5G1). Furthermore, we showed that axonal trafficking and local translation of these mRNAs plays a critical role in the generation of axonal ATP. Using a global gene expression analysis, this study identified a highly diverse population of nuclear-encoded mRNAs that were enriched in the axon and presynaptic nerve terminals. Among this population of mRNAs, fifty seven were found to be at least two-fold more abundant in distal axons, as compared with the parental cell bodies. Gene ontology analysis of the nuclear-encoded mitochondrial mRNAs suggested functions for these gene products in molecular and biological processes, including but not limited to oxidoreductase and electron carrier activity and proton transport. Based on these results, we postulate that local translation of nuclear-encoded mitochondrial mRNAs present in the axons may play an essential role in local energy production and maintenance of mitochondrial function.

  11. Axonal transport disruption in peripheral nerve disease

    PubMed Central

    Lloyd, Thomas E.

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  14. A model of axonal transport drug delivery

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Andrey V.

    2012-04-01

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

  15. Synaptic Democracy and Vesicular Transport in Axons

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Levien, Ethan

    2015-04-01

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

  16. Mitochondrial dysfunction in distal axons contribute to HIV sensory neuropathy

    PubMed Central

    Lehmann, Helmar C.; Chen, Weiran; Borzan, Jasenka; Mankowski, Joseph; Höke, Ahmet

    2010-01-01

    Objective Accumulation of mitochondrial DNA (mtDNA) damage has been associated with aging and abnormal oxidative metabolism. We hypothesized that in human immunodeficiency virus associated sensory neuropathy (HIV-SN), damaged mtDNA accumulates in distal nerve segments and that a spatial pattern of mitochondrial dysfunction contribute to the distal degeneration of sensory nerve fibers. Methods We measured levels of common deletion mutations in mtDNA and expression levels of mitochondrial respiratory chain complexes of matched proximal and distal nerve specimens from patients with and without HIV-SN. In mitochondria isolated from peripheral nerves of simian immunodeficiency virus (SIV) infected macaques, a model of HIV-SN, we measured mitochondrial function and generation of reactive oxygen species. Results We identified increased levels of mtDNA common deletion mutation in post-mortem sural nerves of patients with HIV-SN as compared to uninfected patients or HIV patients without sensory neuropathy. Furthermore, we found that common deletion mutation in mtDNA was more prevalent in distal sural nerves compared to dorsal root ganglia. In a primate model of HIV-SN, freshly isolated mitochondria from sural nerves of macaques infected with a neurovirulent strain of SIV showed impaired mitochondrial function compared to mitochondria from proximal nerve segments. Interpretation Our findings suggest that mtDNA damage accumulates in distal mitochondria of long axons, especially in patients with HIV-SN, and that this may lead to reduced mitochondrial function in distal nerves relative to proximal segments. Although our findings are based on HIV-SN, if confirmed in other neuropathies, these observations could explain the length-dependent nature of most axonal peripheral neuropathies. PMID:21280080

  17. 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

  18. Transport of cytoskeletal elements in the squid giant axon.

    PubMed Central

    Terasaki, M; Schmidek, A; Galbraith, J A; Gallant, P E; Reese, T S

    1995-01-01

    In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon. Images Fig. 1 Fig. 2 Fig. 3 PMID:8524791

  19. Transport of cytoskeletal elements in the squid giant axon.

    PubMed

    Terasaki, M; Schmidek, A; Galbraith, J A; Gallant, P E; Reese, T S

    1995-12-01

    In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. The role of stretching in slow axonal transport.

    PubMed

    O'Toole, Matthew; Miller, Kyle E

    2011-01-19

    Axonal stretching is linked to rapid rates of axonal elongation. Yet the impact of stretching on elongation and slow axonal transport is unclear. Here, we develop a mathematical model of slow axonal transport that incorporates the rate of axonal elongation, protein half-life, protein density, adhesion strength, and axonal viscosity to quantify the effects of axonal stretching. We find that under conditions where the axon (or nerve) is free of a substrate and lengthens at rapid rates (>4 mm day⁻¹), stretching can account for almost 50% of total anterograde axonal transport. These results suggest that it is possible to accelerate elongation and transport simultaneously by increasing either the axon's susceptibility to stretching or the forces that induce stretching. To our knowledge, this work is the first to incorporate the effects of stretching in a model of slow axonal transport. It has relevance to our understanding of neurite outgrowth during development and peripheral nerve regeneration after trauma, and hence to the development of treatments for spinal cord injury.

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

    PubMed

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

    2016-01-01

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

  6. 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

  7. Overexpression of neurofilament subunit M accelerates axonal transport of neurofilaments.

    PubMed

    Xu, Z; Tung, V W

    2000-06-01

    Neurofilaments are composed of three polypeptide subunits (NF-H, NF-M and NF-L). They are the most abundant cytoskeletal element in large myelinated axons and play a central role in development of axonal caliber. To perform this role, neurofilaments are transported from their site of synthesis, the cell bodies, to the distal axons. Previous studies showed that overexpression of NF-M in transgenic mice led to accumulation of neurofilaments in neurons and a reduction in the number of neurofilaments in axons, suggesting that axonal transport of neurofilaments was slowed. To determine whether this was the case, we measured axonal transport velocities in the wild type and transgenic mice overexpressing NF-M by the classical pulse-labeling method using 35S-methionine. We found that neurofilament transport in peripheral motor axons can be described with a model consistent with two linear velocities. Contrary to expectations, both velocities were accelerated by overexpression of NF-M. These results suggest that subunit composition in neurofilaments play a regulatory role in neurofilament transport. In addition, these results show that there are regional differences in neurofilament transport along long axons and these differences may be the basis for selective regional accumulation of neurofilaments in various neurological disorders.

  8. 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

  9. 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).

  10. 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

    2016-07-02

    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.

  11. Axotomy accelerates slow component b of axonal transport.

    PubMed

    Jacob, J M; McQuarrie, I G

    1991-09-01

    Because the integrity of an axon depends on the supply of proteins synthesized in the cell body, we examined the effect of axotomy on the transport of structural proteins in rat motor axons, and the effect of altered transport on the rate of outgrowth after a subsequent testing axotomy. To examine the axonal transport of structural proteins, we labeled newly synthesized proteins with 35S-methionine 7 days after a "conditioning" lesion of the sciatic nerve, and removed the nerve 7-21 days later for SDS-PAGE. Tubulin, actin, calmodulin, and the 68-kD light neurofilament protein (NF-L) were identified by fluorography and removed for liquid scintillation counting. The fastest moving structural proteins were carried by slow component b (SCb) of axonal transport, which advanced 20% faster in conditioned axons: 4.2 versus 3.5 mm/day (p less than 0.01). NF-L was not accelerated, indicating that the motor for subcomponent a (SCa) of slow axonal transport was unaffected by axotomy. To measure outgrowth distances, the testing lesions was made 7 days after the conditioning lesion, and growth cones were located by the fast transport method 3 or 9 days later. The regression analysis of outgrowth distance on time showed that sprouts elongated 25% faster in conditioned axons: 4.0 versus 3.2 mm/day (p less than 0.001). These accelerated sprouts were formed too far from the spinal cord to contain SCb proteins that were synthesized after axotomy. Because the rate of outgrowth correlated closely with the rate of SCb in outgrowing sprouts (McQuarrie and Jacob, J. Comp. Neurol. 305:139-147, 1991), we conclude that SCb is accelerated throughout the length of the axon by 7 days after axotomy.

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

    PubMed

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

    2015-04-01

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

  13. 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

  14. Dissociation of Axonal Neurofilament Content from Its Transport Rate.

    PubMed

    Yuan, Aidong; Hassinger, Linda; Rao, Mala V; Julien, Jean-Pierre; Miller, Christopher C J; Nixon, Ralph A

    2015-01-01

    The axonal cytoskeleton of neurofilament (NF) is a long-lived network of fibrous elements believed to be a stationary structure maintained by a small pool of transported cytoskeletal precursors. Accordingly, it may be predicted that NF content in axons can vary independently from the transport rate of NF. In the present report, we confirm this prediction by showing that human NFH transgenic mice and transgenic mice expressing human NFL Ser55 (Asp) develop nearly identical abnormal patterns of NF accumulation and distribution in association with opposite changes in NF slow transport rates. We also show that the rate of NF transport in wild-type mice remains constant along a length of the optic axon where NF content varies 3-fold. Moreover, knockout mice lacking NFH develop even more extreme (6-fold) proximal to distal variation in NF number, which is associated with a normal wild-type rate of NF transport. The independence of regional NF content and NF transport is consistent with previous evidence suggesting that the rate of incorporation of transported NF precursors into a metabolically stable stationary cytoskeletal network is the major determinant of axonal NF content, enabling the generation of the striking local variations in NF number seen along axons.

  15. 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

  16. Evidence that Myosin Activity Opposes Microtubule-based Axonal Transport of Mitochondria

    PubMed Central

    Pathak, Divya; Sepp, Katharine J.; Hollenbeck, Peter J.

    2010-01-01

    Neurons transport and position mitochondria using a combination of saltatory, bidirectional movements and stationary docking. Axonal mitochondria move along microtubules (MTs) using kinesin and dynein motors, but actin and myosin also play a poorly-defined role in their traffic. To ascertain this role, we have used RNA interference to deplete specific myosin motors in cultured Drosophila neurons and quantified the effects on mitochondrial motility. We produced a fly strain expressing the C. elegans RNA transporter SID-1 in neurons to increase the efficacy of RNAi in primary cultures. These neurons exhibited significantly increased RNAi-mediated knockdown of gene expression compared to neurons not expressing this transporter. Using this system, we observed a significant increase in mitochondrial transport upon myosin V depletion. Mitochondrial mean velocity and duty cycle were augmented in both anterograde and retrograde directions, and the fraction of mitochondrial flux contained in long runs almost doubled for anterograde movement. Myosin VI depletion increased the same movement parameters, but was selective for retrograde movement, while myosin II depletion produced no phenotype. An additional effect of myosin V depletion was an increase in mitochondrial length. These data indicate that myosin V and VI play related but distinct roles in regulating MT-based mitochondrial movement: they oppose, rather than complement protracted MT-based movements and perhaps facilitate organelle docking. PMID:20592219

  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. 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

  19. Loss of mitochondrial fission depletes axonal mitochondria in midbrain dopamine neurons.

    PubMed

    Berthet, Amandine; Margolis, Elyssa B; Zhang, Jue; Hsieh, Ivy; Zhang, Jiasheng; Hnasko, Thomas S; Ahmad, Jawad; Edwards, Robert H; Sesaki, Hiromi; Huang, Eric J; Nakamura, Ken

    2014-10-22

    Disruptions in mitochondrial dynamics may contribute to the selective degeneration of dopamine (DA) neurons in Parkinson's disease (PD). However, little is known about the normal functions of mitochondrial dynamics in these neurons, especially in axons where degeneration begins, and this makes it difficult to understand the disease process. To study one aspect of mitochondrial dynamics-mitochondrial fission-in mouse DA neurons, we deleted the central fission protein dynamin-related protein 1 (Drp1). Drp1 loss rapidly eliminates the DA terminals in the caudate-putamen and causes cell bodies in the midbrain to degenerate and lose α-synuclein. Without Drp1, mitochondrial mass dramatically decreases, especially in axons, where the mitochondrial movement becomes uncoordinated. However, in the ventral tegmental area (VTA), a subset of midbrain DA neurons characterized by small hyperpolarization-activated cation currents (Ih) is spared, despite near complete loss of their axonal mitochondria. Drp1 is thus critical for targeting mitochondria to the nerve terminal, and a disruption in mitochondrial fission can contribute to the preferential death of nigrostriatal DA neurons.

  20. Determination of axonal transport velocities via image cross- and autocorrelation.

    PubMed

    Welzel, Oliver; Boening, Daniel; Stroebel, Armin; Reulbach, Udo; Klingauf, Jurgen; Kornhuber, Johannes; Groemer, Teja Wolfgang

    2009-09-01

    On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR.

  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. 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.

  3. Oxidative damage to mitochondria at the nodes of Ranvier precedes axon degeneration in ex vivo transected axons.

    PubMed

    Bros, Helena; Millward, Jason M; Paul, Friedemann; Niesner, Raluca; Infante-Duarte, Carmen

    2014-11-01

    Oxidative stress and mitochondrial dysfunction appear to contribute to axon degeneration in numerous neurological disorders. However, how these two processes interact to cause axonal damage-and how this damage is initiated-remains unclear. In this study we used transected motor axons from murine peripheral roots to investigate whether oxidative stress alters mitochondrial dynamics in myelinated axons. We show that the nodes of Ranvier are the initial sites of mitochondrial damage induced by oxidative stress. There, mitochondria became depolarized, followed by alterations of the external morphology and disruption of the cristae, along with reduced mitochondrial transport. These mitochondrial changes expanded from the nodes of Ranvier bidirectionally towards both internodes and eventually affected the entire mitochondrial population in the axon. Supplementing axonal bioenergetics by applying nicotinamide adenine dinucleotide and methyl pyruvate, rendered the mitochondria at the nodes of Ranvier resistant to these oxidative stress-induced changes. Importantly, this inhibition of mitochondrial damage protected the axons from degeneration. In conclusion, we present a novel ex vivo approach for monitoring mitochondrial dynamics within axons, which proved suitable for detecting mitochondrial changes upon exogenous application of oxidative stress. Our results indicate that the nodes of Ranvier are the site of initial mitochondrial damage in peripheral axons, and suggest that dysregulation of axonal bioenergetics plays a critical role in oxidative stress-triggered mitochondrial alterations and subsequent axonal injury. These novel insights into the mechanisms underlying axon degeneration may have implications for neurological disorders with a degenerative component.

  4. 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

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

    PubMed Central

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

    2010-01-01

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

  6. Single molecule imaging of NGF axonal transport in microfluidic devices

    PubMed Central

    Zhang, Kai; Osakada, Yasuko; Vrljic, Marija; Chen, Liang; Mudrakola, Harsha V.

    2010-01-01

    Nerve growth factor (NGF) signaling begins at the nerve terminal, where it binds and activates membrane receptors and subsequently carries the cell-survival signal to the cell body through the axon. A recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single molecule imaging an essential tool for NGF studies. Despite being an increasingly popular technique, single molecule imaging in live cells is often limited by background fluorescence. Here, we employed a microfluidic culture platform to achieve background reduction for single molecule imaging in live neurons. Microfluidic devices guide the growth of neurons and allow separately-controlled microenvironment for cell bodies or axon termini. Designs of microfluidic devices were optimized and a three-compartment device successfully achieved direct observation of axonal transport of single NGF when quantum dot labeled NGF (Qdot-NGF) was applied only to the distal-axon compartment while imaging was carried out exclusively in the cell-body compartment. Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36–14°C. A 10°C decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Our successful measurements of NGF transport suggest that the microfluidic device can serve as a unique platform for single molecule imaging of molecular processes in neurons. PMID:20623041

  7. Axonal Transport and the Delivery of Presynaptic Components

    PubMed Central

    Goldstein, Ann Y. N.; Wang, Xinnan

    2008-01-01

    Summary The mechanisms for delivering components to nerve terminals are diverse and highly regulated. The diversity of kinesin motors alone is insufficient to account for the specificity of delivery. Additional specificity and control are contributed by adaptor proteins and associated regulatory molecules. The interaction of cargos with these complexes can confer distinct behaviors on the transport of synaptic organelles. The rich regulatory mechanisms of transport are only now emerging as the cargo-motor complexes are defined and subsequent local events that regulate their dynamic relationship are examined. Here we review recent studies of kinesin-related axonal transport of three crucial synaptic components, Piccolo-bassoon Transport Vesicles (PTVs), Synaptic Vesicle Precursors (SVPs), and mitochondria, and the mechanisms that modulate their transport. PMID:18950710

  8. 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

  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. 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

  11. MIRO GTPases in Mitochondrial Transport, Homeostasis and Pathology.

    PubMed

    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 Ca(2+)-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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Sainath, Rajiv; Gallo, Gianluca

    2015-07-01

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

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

    PubMed

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

    2011-07-01

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

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

    PubMed

    Terada, S; Kinjo, M; Hirokawa, N

    2000-09-29

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

  16. Tau phosphorylation affects its axonal transport and degradation

    PubMed Central

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

    2013-01-01

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

  17. 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.

  18. 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

  19. 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

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

    PubMed

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

    2016-02-15

    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

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

    PubMed

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

    2016-02-15

    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.

  2. UNC-51/ATG1 kinase regulates axonal transport by mediating motor–cargo assembly

    PubMed Central

    Toda, Hirofumi; Mochizuki, Hiroaki; Flores, Rafael; Josowitz, Rebecca; Krasieva, Tatiana B.; LaMorte, Vickie J.; Suzuki, Emiko; Gindhart, Joseph G.; Furukubo-Tokunaga, Katsuo; Tomoda, Toshifumi

    2008-01-01

    Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor–cargo assembly are not well understood. Here we show that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser143, and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor−cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser143 phosphorylation in axonal transport. Thus, our data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins. PMID:19056884

  3. 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.

  4. Neurofilament phosphorylation regulates axonal transport by an indirect mechanism: a merging of opposing hypotheses.

    PubMed

    Shea, Thomas B; Lee, Sangmook

    2011-11-01

    Neurofilaments (NFs) are among the most abundant constituents of the axonal cytoskeleton. NFs consist of four subunits, termed NF-H, NF-M and NF-L, corresponding to heavy, medium and light in reference to their molecular mass and α-internexin. Phosphorylation of the C-terminal "sidearms" of NF-H and NF-M regulates the ability of NFs to form a cytoskeletal lattice that supports the mature axon. C-terminal phosphorylation events have classically been considered to regulate NF axonal transport. By contrast, studies demonstrating that NF axonal transport was not accelerated following sidearm deletion provided evidence that phosphorylation does not regulate NF transport. Herein, we demonstrate how comparison of transport and distribution of differentially phosphorylated NFs along axons identify common ground between these hypotheses and may resolve this controversy.

  5. Axonal mRNA in uninjured and regenerating cortical mammalian axons

    PubMed Central

    Taylor, Anne M.; Berchtold, Nicole C.; Perreau, Victoria M.; Tu, Christina H.; Jeon, Noo Li; Cotman, Carl W.

    2013-01-01

    Using a novel microfluidic chamber that allows the isolation of axons without contamination by non-axonal material, we have for the first time purified mRNA from naïve, matured CNS axons, and identified the presence of >300 mRNA transcripts. We demonstrate that the transcripts are axonal in nature, and that many of the transcripts present in uninjured CNS axons overlap with those previously identified in PNS injury-conditioned DRG axons. The axonal transcripts detected in matured cortical axons are enriched for protein translational machinery, transport, cytoskeletal components, and mitochondrial maintenance. We next investigated how the axonal mRNA pool changes after axotomy, revealing that numerous gene transcripts related to intracellular transport, mitochondria and the cytoskeleton show decreased localization two days after injury. In contrast, gene transcripts related to axonal targeting and synaptic function show increased localization in regenerating cortical axons, suggesting that there is an increased capacity for axonal outgrowth and targeting, and increased support for synapse formation and presynaptic function in regenerating CNS axons after injury. Our data demonstrate that CNS axons contain many mRNA species of diverse functions, and suggest that, like invertebrate and PNS axons, CNS axons synthesize proteins locally, maintaining a degree of autonomy from the cell body. PMID:19369540

  6. Myelination and axonal electrical activity modulate the distribution and motility of mitochondria at CNS nodes of Ranvier.

    PubMed

    Ohno, Nobuhiko; Kidd, Grahame J; Mahad, Don; Kiryu-Seo, Sumiko; Avishai, Amir; Komuro, Hitoshi; Trapp, Bruce D

    2011-05-18

    Energy production presents a formidable challenge to axons as their mitochondria are synthesized and degraded in neuronal cell bodies. To meet the energy demands of nerve conduction, small mitochondria are transported to and enriched at mitochondrial stationary sites located throughout the axon. In this study, we investigated whether size and motility of mitochondria in small myelinated CNS axons are differentially regulated at nodes, and whether mitochondrial distribution and motility are modulated by axonal electrical activity. The size/volume of mitochondrial stationary sites was significantly larger in juxtaparanodal/internodal axoplasm than in nodal/paranodal axoplasm. With three-dimensional electron microscopy, we observed that axonal mitochondrial stationary sites were composed of multiple mitochondria of varying length, except at nodes where mitochondria were uniformly short and frequently absent altogether. Mitochondrial transport speed was significantly reduced in nodal axoplasm compared with internodal axoplasm. Increased axonal electrical activity decreased mitochondrial transport and increased the size of mitochondrial stationary sites in nodal/paranodal axoplasm. Decreased axonal electrical activity had the opposite effect. In cerebellar axons of the myelin-deficient rat, which contain voltage-gated Na(+) channel clusters but lack paranodal specializations, axonal mitochondrial motility and stationary site size were similar at Na(+) channel clusters and other axonal regions. These results demonstrate juxtaparanodal/internodal enrichment of stationary mitochondria and neuronal activity-dependent dynamic modulation of mitochondrial distribution and transport in nodal axoplasm. In addition, the modulation of mitochondrial distribution and motility requires oligodendrocyte-axon interactions at paranodal specializations. PMID:21593309

  7. Dual Role of Herpes Simplex Virus 1 pUS9 in Virus Anterograde Axonal Transport and Final Assembly in Growth Cones in Distal Axons

    PubMed Central

    Boadle, Ross A.

    2015-01-01

    ABSTRACT The herpes simplex virus type 1 (HSV-1) envelope protein pUS9 plays an important role in virus anterograde axonal transport and spread from neuronal axons. In this study, we used both confocal microscopy and transmission electron microscopy (TEM) to examine the role of pUS9 in the anterograde transport and assembly of HSV-1 in the distal axon of human and rat dorsal root ganglion (DRG) neurons using US9 deletion (US9−), repair (US9R), and wild-type (strain F, 17, and KOS) viruses. Using confocal microscopy and single and trichamber culture systems, we observed a reduction but not complete block in the anterograde axonal transport of capsids to distal axons as well as a marked (∼90%) reduction in virus spread from axons to Vero cells with the US9 deletion viruses. Axonal transport of glycoproteins (gC, gD, and gE) was unaffected. Using TEM, there was a marked reduction or absence of enveloped capsids, in varicosities and growth cones, in KOS strain and US9 deletion viruses, respectively. Capsids (40 to 75%) in varicosities and growth cones infected with strain 17, F, and US9 repair viruses were fully enveloped compared to less than 5% of capsids found in distal axons infected with the KOS strain virus (which also lacks pUS9) and still lower (<2%) with the US9 deletion viruses. Hence, there was a secondary defect in virus assembly in distal axons in the absence of pUS9 despite the presence of key envelope proteins. Overall, our study supports a dual role for pUS9, first in anterograde axonal transport and second in virus assembly in growth cones in distal axons. IMPORTANCE HSV-1 has evolved mechanisms for its efficient transport along sensory axons and subsequent spread from axons to epithelial cells after reactivation. In this study, we show that deletion of the envelope protein pUS9 leads to defects in virus transport along axons (partial defect) and in virus assembly and egress from growth cones (marked defect). Virus assembly and exit in the neuronal

  8. 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

  9. Axonal transport of neurofilament is accelerated in peripheral nerve during 2,5-hexanedione intoxication.

    PubMed

    Monaco, S; Jacob, J; Jenich, H; Patton, A; Autilio-Gambetti, L; Gambetti, P

    1989-07-10

    The neurotoxic compound 2,5-hexanedione (2,5-HD) causes an axonopathy characterized by the presence of neurofilament (NF)-containing enlargements in the preterminal segments of central and peripheral axons. The 2,5-HD axonopathy is a good model for human acquired and inherited giant axonal neuropathies. Recently, we reported that following 2,5-HD administration, axonal transport of NF is markedly and selectively accelerated in the primary visual system. We have now studied slow axonal transport in the sciatic system of rats intoxicated with 0.5% 2,5-HD in drinking water. Following radiolabeling, transported proteins were examined after polyacrylamide gel electrophoresis and fluorography. The bulk of radiolabeled NF subunits was located 30-50 mm from the spinal cord in 2,5-HD treated animals and 10-25 mm in controls. The rate of transport of the three NF subunits was 0.7 mm/day in controls and 1.2 mm/day in 2,5-HD treated animals. The rate of transport of tubulin was not significantly changed. Electrophysiological studies of soleus nerve and muscle showed no evidence of denervation after 6 weeks of intoxication. It is concluded that, following 2,5-HD administration, transport of NF is preferentially accelerated in both central and peripheral axons. A pathogenetic mechanism based on the acceleration of NF transport is proposed, which may explain the formation and the distal or proximal location of NF-containing axonal enlargements in giant axonopathies.

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

    PubMed

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

    2009-04-01

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

  11. Enhanced Transcriptional Activity and Mitochondrial Localization of STAT3 Co-induce Axon Regrowth in the Adult Central Nervous System.

    PubMed

    Luo, Xueting; Ribeiro, Marcio; Bray, Eric R; Lee, Do-Hun; Yungher, Benjamin J; Mehta, Saloni T; Thakor, Kinjal A; Diaz, Francisca; Lee, Jae K; Moraes, Carlos T; Bixby, John L; Lemmon, Vance P; Park, Kevin K

    2016-04-12

    Signal transducer and activator of transcription 3 (STAT3) is a transcription factor central to axon regrowth with an enigmatic ability to act in different subcellular regions independently of its transcriptional roles. However, its roles in mature CNS neurons remain unclear. Here, we show that along with nuclear translocation, STAT3 translocates to mitochondria in mature CNS neurons upon cytokine stimulation. Loss- and gain-of-function studies using knockout mice and viral expression of various STAT3 mutants demonstrate that STAT3's transcriptional function is indispensable for CNS axon regrowth, whereas mitochondrial STAT3 enhances bioenergetics and further potentiates regrowth. STAT3's localization, functions, and growth-promoting effects are regulated by mitogen-activated protein kinase kinase (MEK), an effect further enhanced by Pten deletion, leading to extensive axon regrowth in the mouse optic pathway and spinal cord. These results highlight CNS neuronal dependence on STAT3 transcriptional activity, with mitochondrial STAT3 providing ancillary roles, and illustrate a critical contribution for MEK in enhancing diverse STAT3 functions and axon regrowth.

  12. 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

  13. Signalling endosomes in axonal transport: travel updates on the molecular highway.

    PubMed

    Schmieg, Nathalie; Menendez, Guillermo; Schiavo, Giampietro; Terenzio, Marco

    2014-03-01

    Neurons are highly polarised cells. They make contact with their targets through long axons, along which a steady flux of proteins, lipids, nucleic acids and organelles is constantly maintained. This process is crucial to the development and maintenance of the nervous system, as proven by the many neurodegenerative disorders associated with defective axonal transport. Specific pools of endocytic organelles, which travel along the axon towards the cell body, have assumed a growing importance by virtue of their transported signals. These organelles, named signalling endosomes, vehicle growth factors, such as neurotrophins, and their signalling receptors all the way from the axon terminals to the neuronal cell body. Due to the central importance of neurotrophins in neuronal development and survival, significant efforts have gone over the years into the study of long-range neutrophin trafficking and signalling. Recent evidence has pointed to a role of signalling endosomes in the axonal retrograde transport of many morphogenetic and survival factors, increasing their importance even further. In light of these findings, signalling endosomes have shown potential for integration of different growth factors signals and the ability to decode them by differential sorting in the neuronal cell body. In this review we aim to discuss the state of the field regarding the nature and dynamics of signalling endosomes, their signalling capabilities, their energy requirements for axonal transport and last but not least, their importance in health and disease.

  14. Difference Tracker: ImageJ plugins for fully automated analysis of multiple axonal transport parameters.

    PubMed

    Andrews, Simon; Gilley, Jonathan; Coleman, Michael P

    2010-11-30

    Studies of axonal transport are critical, not only to understand its normal regulation, but also to determine the roles of transport impairment in disease. Exciting new resources have recently become available allowing live imaging of axonal transport in physiologically relevant settings, such as mammalian nerves. Thus the effects of disease, ageing and therapies can now be assessed directly in nervous system tissue. However, these imaging studies present new challenges. Manual or semi-automated analysis of the range of transport parameters required for a suitably complete evaluation is very time-consuming and can be subjective due to the complexity of the particle movements in axons in ex vivo explants or in vivo. We have developed Difference Tracker, a program combining two new plugins for the ImageJ image-analysis freeware, to provide fast, fully automated and objective analysis of a number of relevant measures of trafficking of fluorescently labeled particles so that axonal transport in different situations can be easily compared. We confirm that Difference Tracker can accurately track moving particles in highly simplified, artificial simulations. It can also identify and track multiple motile fluorescently labeled mitochondria simultaneously in time-lapse image stacks from live imaging of tibial nerve axons, reporting values for a number of parameters that are comparable to those obtained through manual analysis of the same axons. Difference Tracker therefore represents a useful free resource for the comparative analysis of axonal transport under different conditions, and could potentially be used and developed further in many other studies requiring quantification of particle movements.

  15. In vitro low frequency electromagnetic field effect on fast axonal transport.

    PubMed

    Zborowski, M; Atkinson, M; Lewandowski, J J; Jacobs, G; Mitchell, D; Breuer, A C; Nosé, Y

    1988-01-01

    The objective of this study was to evaluate the effects of a low frequency electromagnetic field on fast axonal transport for future neuroprosthetic applications. Changes in speeds and densities of retrograde fast organelle transport in rat sciatic nerve preparations were measured in vitro upon exposure to 15 and 50 Hz pulsed magnetic fields with peak intensities of 4.4 and 8.8 mT. Maximum current density of the induced eddy current was calculated to be about 40 microA/cm2. Video enhanced differential interference contrast microscopy was used to record axons supporting active organelle transport. Strong effects were observed in myelinated axons (cessation of transport in up to 10 min). Such effects may eventually be used as part of a neuroprosthesis to noninvasively modify or couple to various parts of the nervous system.

  16. 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.

  17. Cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons.

    PubMed

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

    2011-12-01

    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

  18. 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

  19. Axonal autophagosomes use the ride-on service for retrograde transport toward the soma.

    PubMed

    Cheng, Xiu-Tang; Zhou, Bing; Lin, Mei-Yao; Cai, Qian; Sheng, Zu-Hang

    2015-01-01

    Degradation of autophagic vacuoles (AVs) via lysosomes is an important homeostatic process in cells. Neurons are highly polarized cells with long axons, thus facing special challenges to transport AVs generated at distal processes toward the soma where mature acidic lysosomes are relatively enriched. We recently revealed a new motor-adaptor sharing mechanism driving autophagosome transport to the soma. Late endosome (LE)-loaded dynein-SNAPIN motor-adaptor complexes mediate the retrograde transport of autophagosomes upon their fusion with LEs in distal axons. This motor-adaptor sharing mechanism enables neurons to maintain effective autophagic clearance in the soma, thus reducing autophagic stress in axons. Therefore, our study reveals a new cellular mechanism underlying the removal of distal AVs engulfing aggregated misfolded proteins and dysfunctional organelles associated with several major neurodegenerative diseases.

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed

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

    1999-09-28

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

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

    PubMed Central

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

    2010-01-01

    Precise targeting of various voltage-gated ion channels to proper membrane domains is crucial for their distinct roles in neuronal excitability and synaptic transmission. How each channel protein is transported within the cytoplasm is poorly understood. Here we report that KIF5/kinesin I transports Kv3.1 voltage-gated K+ (Kv) channels through the axon initial segment (AIS) via direct binding. First, we have identified a novel interaction between Kv3.1 and KIF5, confirmed by immunoprecipitation from mouse brain lysates and by pull down assays with exogenously-expressed proteins. The interaction is mediated by a direct binding between the Kv3.1 N-terminal T1 domain and a conserved region in KIF5 tail domains, in which proper T1 tetramerization is crucial. Over-expression of this region of KIF5B markedly reduces axonal levels of Kv3.1bHA. In mature hippocampal neurons, endogenous Kv3.1b and KIF5 colocalize. Suppressing the endogenous KIF5B level by siRNA significantly reduces the Kv3.1b axonal level. Furthermore, mutating the Zn2+-binding site within T1 markedly decreases channel axonal targeting and forward trafficking, likely through disrupting T1 tetramerization and hence eliminating the binding to KIF5 tail. The mutation also alters channel activity. Interestingly, co-expression of the YFP-tagged KIF5B assists dendritic Kv3.1a and even mutants with a faulty axonal targeting motif to penetrate the AIS. Finally, fluorescently tagged Kv3.1 channels co-localize and co-move with KIF5B along axons revealed by two-color time-lapse imaging. Our findings suggest that the binding to KIF5 ensures properly assembled and functioning Kv3.1 channels to be transported into axons. PMID:21106837

  3. 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

  4. 3,4-Dimethyl-2,5-hexanedione impairs the axonal transport of neurofilament proteins.

    PubMed

    Griffin, J W; Anthony, D C; Fahnestock, K E; Hoffman, P N; Graham, D G

    1984-06-01

    Accumulations of neurofilaments are observed in a variety of neurological disorders, and their pathogenesis is a fundamental problem of neuropathology. 2,5-Hexanedione (HD) neurotoxicity provides an extensively studied model of axonal neurofibrillary changes in which the pathogenetic mechanisms have been conjectural. Chronic exposure to HD results in neurofilament-filled swellings in the distal regions of large axons of exposed humans and experimental animals. In this report we describe the changes produced by a potent analogue of HD, 3,4-dimethyl-2,5-hexanedione ( DMHD ), in slow axonal transport in the rat sciatic motor axons. Young rats received 0.6 mmol/kg of DMHD for 5 days before [35S]methionine was injected into the lumbar ventral horns. Slow axonal transport of the neurofilament proteins, tubulin, and selected slow component b (SCb) proteins in DMHD -treated animals was compared to the profiles found in age-matched control animals. DMHD administration reduced the rate of transport of the neurofilament proteins 75 to 90%, while tubulin and the SCb proteins were only modestly retarded. No alterations in electrophoretic mobilities of slowly transported proteins were found, nor were any proteins accelerated in transport. These findings were systematically compared to the changes produced by administration of beta,beta'- immino - dipropionitrile (IDPN) (2.0 gm/kg, i.p.), an agent known to impair neurofilament transport. Although slightly less severe, the changes produced by DMHD were nearly identical to those of IDPN. In correlative morphological studies, the neurofilamentous changes were also comparable. The results indicate that DMHD and IDPN share the capacity to interfere selectively with neurofilament transport and thereby share pathogenetic mechanisms. DMHD provides a new agent for exploration of the organization and transport of the neuronal cytoskeleton.

  5. 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

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

    PubMed Central

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

    2012-01-01

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

  7. 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

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

    PubMed

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

    2014-04-01

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

  9. 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

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

    PubMed

    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

    2014-06-01

    In Krabbe's disease (KD), a leukodystrophy caused by β-galactosylceramidase deficiency, demyelination and a myelin-independent axonopathy contributes 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 in the 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 time point 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, opens new windows of action to effectively correct the neuropathology that characterizes this disorder.

  11. 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…

  12. 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.

  13. Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events.

    PubMed

    Motil, Jennifer; Chan, Walter K-H; Dubey, Maya; Chaudhury, Pulkit; Pimenta, Aurea; Chylinski, Teresa M; Ortiz, Daniela T; Shea, Thomas B

    2006-05-01

    We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.

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

    PubMed

    Langford, G M

    1999-12-01

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

  15. Making the case: Married versus Separate models of alphaherpes virus anterograde transport in axons

    PubMed Central

    Kratchmarov, R.; Taylor, M. P.; Enquist, L. W.

    2012-01-01

    SUMMARY Alphaherpesvirus virions infect neurons and are transported in axons for long distance spread within the host nervous system. The assembly state of newly made herpesvirus particles during anterograde transport in axons is an essential question in alphaherpesvirus biology. The structure of the particle has remained both elusive and controversial for the past two decades, with conflicting evidence from EM, immunofluorescence, and live cell imaging studies. Two opposing models have been proposed—the Married and Separate Models. Under the Married Model, infectious virions are assembled in the neuronal cell body before sorting into axons and then traffic inside a transport vesicle. Conversely, the Separate Model postulates that vesicles containing viral membrane proteins are sorted into axons independent of capsids, with final assembly of mature virions occurring at a distant egress site. Recently, a complementary series of studies employing high-resolution EM and live cell fluorescence microscopy have provided evidence consistent with the Married Model, whereas other studies offer evidence supporting the Separate Model. In this review, we compare and discuss the published data and attempt to reconcile divergent findings and interpretations as they relate to these models. PMID:22807192

  16. 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).

  17. 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

  18. 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

  19. Effects of beta 2 adrenergic agonists on axonal injury and mitochondrial metabolism in experimental autoimmune encephalomyelitis rats.

    PubMed

    Zhang, Z W; Qin, X Y; Che, F Y; Xie, G; Shen, L; Bai, Y Y

    2015-01-01

    The primary aims of this study were to investigate mitochondrial metabolism during experimental allergic encephalomyelitis (EAE) animal model axonal injury and to determine the correlation among neurological function scores, pathological changes, and the activities of the BB isoenzyme of creatine kinase (CK-BB), catalase (CAT), and calpain in the brain tissues of EAE rats. Another goal was to preliminarily define the mechanism of mitochondrial metabolism resulting from the effect of beta 2 adrenergic agonists in the process of EAE animal model axonal damage. EAE was induced in specific pathogen free Wistar rats by guinea pig spinal cord homogenate, complete Freund's adjuvant, and pertussis vaccine. We recorded the behavioral change in EAE rats, detected pathological changes in central nervous tissue, and observed the changes of the CK-BB, CAT, and calpain in the EAE rat brain and spinal cord. The results indicated that the average neurologic function score increased in the EAE group compared to that of the controls (P < 0.01). In addition, CAT and CK-BB activities significantly decreased and the calpain activity significantly increased compared with those of the control group (P < 0.05). The decrease of the activity of central nervous CK-BB and CAT content, as well as the increase of calpain activity at the highest time point were considered to be the consequences of EAE. Furthermore, the results revealed that use of salbutamol could alleviate disease symptoms and reduce the recurrence of the EAE disease. PMID:26535670

  20. 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

  1. Carbon disulfide axonopathy. Another experimental model characterized by acceleration of neurofilament transport and distinct changes of axonal size.

    PubMed

    Pappolla, M; Penton, R; Weiss, H S; Miller, C H; Sahenk, Z; Autilio-Gambetti, L; Gambetti, P

    1987-10-27

    The role of axonal transport in the development of structural changes of axons can be examined using experimental models. Two different compounds, 2,5-hexanedione (2,5-HD) and carbon disulfide (CS2), cause axonopathies characterized by the formation of neurofilaments (NF) containing enlargements in preterminal regions of central and peripheral axons. These axonopathies are excellent experimental models of the giant axonal neuropathies, a group of acquired and inherited human diseases of the central and peripheral nervous system. We previously reported that following administration of 2,5-HD, transport of NF is accelerated while number of NF and cross-sectional area are decreased in regions of the axon proximal to the enlargements. We proposed that acceleration of NF transport leads to a 'longitudinal' redistribution of NF which are decreased proximally and increased distally where they form the NF containing axonal enlargements. We have now carried out morphometric, transport and immunocytochemical studies in primary visual axons of rats exposed to CS2. NF-containing axonal enlargements were observed in optic tract and superior colliculus and they increased in number in a proximodistal direction. There was no detectable axonal degeneration and the cross-sectional area of axons proximal to the enlargements was decreased. Transport of NF was markedly accelerated. Immunostaining showed that all 3 NF subunits and phosphorylated epitopes of the 200-kDa NF subunit were present in the NF-containing axonal enlargements. All these findings were similar to those previously observed in the 2,5-HD axonopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

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

    2015-02-19

    The establishment of neuronal polarity is necessary for proper neuronal wiring. Semaphorin3A (Sema3A), originally identified as a repulsive axon guidance molecule, exerts a wide variety of biological functions through signaling pathways including sequential phosphorylation of collapsin response mediator protein by cyclin-dependent kinase-5 (Cdk5) and glycogen synthase kinase-3β (GSK3β). Sema3A acts on its receptor neuropilin-1 to regulate axonal transport. To delineate mechanism by which Sema3A induces axonal transport, we investigate whether GSK3β is involved in mediating Sema3A-induced axonal transport. 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, an inhibitor of GSK3β, suppressed Sema3A-induced antero- and retrograde axonal transport. Introduction of either GSK3β mutants, GSK3β-L128A or K85M, suppressed Sema3A-induced axonal transport. On the other hand, introduction of GSK3β-R96A did not affect the Sema3A effect, suggesting that unprimed substrates are primarily involved in Sema3A-induced axonal transport. Overexpression of a partial fragment of frequently rearranged in advanced T-cell lymphomas 1 (FRATtide), which interferes the interaction between GSK3β and Axis inhibitor-1 (Axin-1), also suppressed Sema3A-induced transport. siRNA knockdown of Axin-1, an unprimed substrate of GSK3β, suppressed Sema3A-induced antero- and retrograde axonal transport. These results indicate that GSK3β and Axin-1 are involved in Sema3A-induced bidirectional axonal transport. This finding should provide a clue for understanding of mechanisms of a wide variety of biological activities of Sema3A. PMID:25528666

  3. Giant axonopathy characterized by intermediate location of axonal enlargements and acceleration of neurofilament transport.

    PubMed

    Monaco, S; Wongmongkolrit, T; Shearson, C M; Patton, A; Schaetzle, B; Autilio-Gambetti, L; Gambetti, P; Sayre, L M

    1990-06-11

    It has previously been shown that 2,5-hexanedione (2,5-HD) and its 3,4-dimethyl derivative (3,4-DMHD) induce neurofilamentous accumulations at prenodal sites in distal and proximal, respectively, regions of peripheral axons. For 2,5-HD, neurofilament (NF) transport is accelerated and this is thought to be directly related to the appearance of the axonal enlargements. For 3,4-DMHD, however, the rate of NF transport cannot be assessed owing to the very proximal position of NF accumulation. In the present study, it is shown that administration to rats of 3-methyl-2,5-hexanedione, the structural 'average' of 2,5-HD and 3,4-DMHD, induces NF accumulations at midway axonal positions of the sciatic and optic systems, and results in acceleration of NF in the sections of optic axons proximal to the enlargements. These results suggest that a common mechanism underlies all gamma-diketone neuropathies, and that the proximodistal pattern of axonal enlargements represents pharmacokinetic variables rather than differences in mode of action. The neurotoxicity of gamma-diketones probably arises from pyrrolation of lysine epsilon-amino groups in crucial regions of NF or related proteins responsible for maintaining the proper supramolecular organization of the cytoskeleton. Acceleration of NF transport appears to be a common characteristic of chemically induced axonopathies, regardless of location, and this is contrary to the theory that gamma-diketone-induced NF accumulation results primarily from a progressive cross-linking of NF occurring subsequent to pyrrole formation.

  4. 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.

  5. Presenilin influences glycogen synthase kinase-3 β (GSK-3β) for kinesin-1 and dynein function during axonal transport.

    PubMed

    Dolma, Kunsang; Iacobucci, Gary J; Hong Zheng, Kan; Shandilya, Jayasha; Toska, Eneda; White, Joseph A; Spina, Elizabeth; Gunawardena, Shermali

    2014-03-01

    Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimer's disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3β (GSK-3β). PS genetically interacts with GSK-3β in an activity-dependent manner. Excess of active GSK-3β perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3β-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3β did not affect axonal transport. Strikingly, GSK-3β-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3β are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3β activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.

  6. 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

  7. Axonal autophagosomes recruit dynein for retrograde transport through fusion with late endosomes.

    PubMed

    Cheng, Xiu-Tang; Zhou, Bing; Lin, Mei-Yao; Cai, Qian; Sheng, Zu-Hang

    2015-05-11

    Efficient degradation of autophagic vacuoles (AVs) via lysosomes is an important cellular homeostatic process. This is particularly challenging for neurons because mature acidic lysosomes are relatively enriched in the soma. Although dynein-driven retrograde transport of AVs was suggested, a fundamental question remains how autophagosomes generated at distal axons acquire dynein motors for retrograde transport toward the soma. In this paper, we demonstrate that late endosome (LE)-loaded dynein-snapin complexes drive AV retrograde transport in axons upon fusion of autophagosomes with LEs into amphisomes. Blocking the fusion with syntaxin17 knockdown reduced recruitment of dynein motors to AVs, thus immobilizing them in axons. Deficiency in dynein-snapin coupling impaired AV transport ,: resulting in AV accumulation in neurites and synaptic terminals. Altogether, our study provides the first evidence that autophagosomes recruit dynein through fusion with LEs and reveals a new motor-adaptor sharing mechanism by which neurons may remove distal AVs engulfing aggregated proteins and dysfunctional organelles for efficient degradation in the soma.

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

    SciTech Connect

    Snyder, R.E.

    1988-04-01

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

  9. 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.

  10. Inhibition of fast axonal transport in vitro by the local anesthetics prilocaine, mepivacaine, and bupivacaine.

    PubMed

    Lavoie, P A

    1983-12-01

    The aim of the present study was to establish the concentrations of prilocaine, mepivacaine, and bupivacaine which are effective at blocking fast axonal transport, to determine whether prilocaine and mepivacaine offer a better prospect of dissociating conduction block and transport block in vivo than does lidocaine and whether bupivacaine offers a better prospect than etidocaine in the same context. Fast axonal transport of [3H]leucine-labeled proteins was studied in vitro in bullfrog spinal nerves and quantitated by liquid scintillation counting. Exposure of spinal nerves to 14 mM prilocaine reduced the quantity of 3H-labeled proteins which accumulated at a ligature by 86%, and exposure to 14 mM mepivacaine reduced it by 70%; 10 mM prilocaine reduced this same parameter by 54%, a degree of inhibition close to the 44% reduction caused by 14 mM lidocaine. The D(-) and L(+) stereoisomers of mepivacaine each reduced transport to the ligature by approximately 50% at a concentration of 14 mM. Bupivacaine reduced the accumulation of 3H-labeled proteins at the ligature by 49% at a 10 mM concentration (pH 6.2); its potency is close to that found for etidocaine in a previous study. Since prilocaine and mepivacaine are at least as potent as lidocaine as transport inhibitors and at blocking impulse conduction, these two anesthetics offer no advantage over lidocaine to achieve dissociation of conduction block from transport block in vivo. Bupivacaine appears to offer no advantage over etidocaine in the same context, as the two agents have a similar potency as local anesthetics and a similar potency as inhibitors of fast axonal transport.

  11. 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

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

    PubMed Central

    1995-01-01

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

  13. 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

  14. MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons.

    PubMed

    Mandelkow, Eva-Maria; Thies, Edda; Trinczek, Bernhard; Biernat, Jacek; Mandelkow, Eckard

    2004-10-11

    Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.

  15. 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

  16. Syntabulin-kinesin-1 family member 5B-mediated axonal transport contributes to activity-dependent presynaptic assembly.

    PubMed

    Cai, Qian; Pan, Ping-Yue; Sheng, Zu-Hang

    2007-07-01

    The mechanism by which microtubule-based axonal transport regulates activity-dependent presynaptic plasticity in developing neurons remains mostly unknown. Our previous studies established that syntabulin is an adaptor capable of conjoining the kinesin family member 5B (KIF5B) motor and syntaxin-1. We now report that the complex of syntaxin-1-syntabulin-KIF5B mediates axonal transport of the active zone (AZ) components essential for presynaptic assembly. Syntabulin associates with AZ precursor carriers and colocalizes and comigrates with green fluorescent protein (GFP)-Bassoon-labeled AZ transport cargos within developing axons. Knock-down of syntabulin or disruption of the syntaxin-1-syntabulin-KIF5B complex impairs the anterograde transport of GFP-Bassoon out of the soma and reduces the axonal densities of synaptic vesicle (SV) clusters and FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-dibutylaminostyryl)pyridinium, dibromide] loading. Furthermore, syntabulin loss of function results in a reduction in both the amplitude of postsynaptic currents and the frequency of asynchronous quantal events, and abolishes the activity-induced recruitment of new GFP-Bassoon into the axons and subsequent coclustering with SVs. Consequently, syntabulin loss of function blocks the formation of new presynaptic boutons during activity-dependent synaptic plasticity in developing neurons. These studies establish that a kinesin motor-adaptor complex is critical for the anterograde axonal transport of AZ components, thus contributing to activity-dependent presynaptic assembly during neuronal development.

  17. In silico aided thoughts on mitochondrial vitamin C transport.

    PubMed

    Szarka, András; Balogh, Tibor

    2015-01-21

    The huge demand of mitochondria as the quantitatively most important sources of ROS in the majority of heterotrophic cells for vitamin C is indisputable. The reduced form of the vitamin, l-ascorbic acid, is imported by an active mechanism requiring two sodium-dependent vitamin C transporters (SVCT1 and SVCT2). The oxidized form, dehydroascorbate is taken up by different members of the GLUT family. Because of the controversial experimental results the picture on mitochondrial vitamin C transport became quite obscure by the spring of 2014. Thus in silico prediction tools were applied in aid of the support of in vitro and in vivo results. The role of GLUT1 as a mitochondrial dehydroascorbate transporter could be reinforced by in silico predictions however the mitochondrial presence of GLUT10 is not likely since this transport protein got far the lowest mitochondrial localization scores. Furthermore the possible roles of GLUT9 and 11 in mitochondrial vitamin C transport can be proposed leastwise on the base of their computational localization analysis. In good concordance with the newest experimental observations on SVCT2 the mitochondrial presence of this transporter could also be supported by the computational prediction tools.

  18. In silico aided thoughts on mitochondrial vitamin C transport.

    PubMed

    Szarka, András; Balogh, Tibor

    2015-01-21

    The huge demand of mitochondria as the quantitatively most important sources of ROS in the majority of heterotrophic cells for vitamin C is indisputable. The reduced form of the vitamin, l-ascorbic acid, is imported by an active mechanism requiring two sodium-dependent vitamin C transporters (SVCT1 and SVCT2). The oxidized form, dehydroascorbate is taken up by different members of the GLUT family. Because of the controversial experimental results the picture on mitochondrial vitamin C transport became quite obscure by the spring of 2014. Thus in silico prediction tools were applied in aid of the support of in vitro and in vivo results. The role of GLUT1 as a mitochondrial dehydroascorbate transporter could be reinforced by in silico predictions however the mitochondrial presence of GLUT10 is not likely since this transport protein got far the lowest mitochondrial localization scores. Furthermore the possible roles of GLUT9 and 11 in mitochondrial vitamin C transport can be proposed leastwise on the base of their computational localization analysis. In good concordance with the newest experimental observations on SVCT2 the mitochondrial presence of this transporter could also be supported by the computational prediction tools. PMID:25451960

  19. 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

  20. 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

  1. 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

  2. Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics.

    PubMed

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

    2015-06-26

    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 Ca(2+) uptake, these include activation of countertransporters (Na(+)/Ca(2+) exchanger and Na(+)/H(+) exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca(2+) binding to matrix buffers. Inorganic phosphate (Pi) is known to affect both the Ca(2+) uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca(2+) dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca(2+) and mitochondrial membrane potential (ΔΨm) to examine the effects of anion transport on mitochondrial Ca(2+) flux and buffering in Pi-depleted guinea pig cardiac mitochondria. Mitochondrial Ca(2+) uptake proceeded slowly in the absence of Pi but matrix free Ca(2+) ([Ca(2+)]mito) still rose to ~50 μm. Pi (0.001-1 mm) accelerated Ca(2+) uptake but decreased [Ca(2+)]mito by almost 50% while restoring ΔΨm. Pi-dependent effects on Ca(2+) were blocked by inhibiting the phosphate carrier. Mitochondrial Ca(2+) uptake rate was also increased by vanadate (Vi), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca(2+) buffering and ΔΨm recovery. Interestingly, ATP or AMP-PNP prevented the effects of Pi on Ca(2+) uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca(2+) uptake and modifies the [Ca(2+)]mito response in a complex manner. PMID:25963147

  3. 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

  4. 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

  5. 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

  6. 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.

  7. Neuronal growth cones respond to laser-induced axonal damage

    PubMed Central

    Wu, Tao; Mohanty, Samarendra; Gomez-Godinez, Veronica; Shi, Linda Z.; Liaw, Lih-Huei; Miotke, Jill; Meyer, Ronald L.; Berns, Michael W.

    2012-01-01

    Although it is well known that damage to neurons results in release of substances that inhibit axonal growth, release of chemical signals from damaged axons that attract axon growth cones has not been observed. In this study, a 532 nm 12 ns laser was focused to a diffraction-limited spot to produce site-specific damage to single goldfish axons in vitro. The axons underwent a localized decrease in thickness (‘thinning’) within seconds. Analysis by fluorescence and transmission electron microscopy indicated that there was no gross rupture of the cell membrane. Mitochondrial transport along the axonal cytoskeleton immediately stopped at the damage site, but recovered over several minutes. Within seconds of damage nearby growth cones extended filopodia towards the injury and were often observed to contact the damaged site. Turning of the growth cone towards the injured axon also was observed. Repair of the laser-induced damage was evidenced by recovery of the axon thickness as well as restoration of mitochondrial movement. We describe a new process of growth cone response to damaged axons. This has been possible through the interface of optics (laser subcellular surgery), fluorescence and electron microscopy, and a goldfish retinal ganglion cell culture model. PMID:21831892

  8. 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

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

    PubMed

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

    2012-03-15

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

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

    PubMed Central

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

    2012-01-01

    Summary Bidirectional transport is a key issue in cellular biology. It requires coordination between microtubule-associated molecular motors that work in opposing directions. The major retrograde and anterograde motors involved in bidirectional transport are cytoplasmic dynein and conventional kinesin, respectively. It is clear that failures in molecular motor activity bear severe consequences, especially in the nervous system. Neuronal migration may be impaired during brain development, and impaired molecular motor activity in the adult is one of the hallmarks of neurodegenerative diseases leading to neuronal cell death. The mechanisms that regulate or coordinate kinesin and dynein activity to generate bidirectional transport of the same cargo are of utmost importance. We examined how Ndel1, a cytoplasmic dynein binding protein, may regulate non-vesicular bidirectional transport. Soluble Ndel1 protein, Ndel1-derived peptides or control proteins were mixed with fluorescent beads, injected into the squid giant axon, and the bead movements were recorded using time-lapse microscopy. Automated tracking allowed for extraction and unbiased analysis of a large data set. Beads moved in both directions with a clear bias to the anterograde direction. Velocities were distributed over a broad range and were typically slower than those associated with fast vesicle transport. Ironically, the main effect of Ndel1 and its derived peptides was an enhancement of anterograde motion. We propose that they may function primarily by inhibition of dynein-dependent resistance, which suggests that both dynein and kinesin motors may remain engaged with microtubules during bidirectional transport. PMID:23213412

  11. 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

  12. 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

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

    PubMed

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

    2015-12-10

    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.

  14. Pathogenesis of herpetic neuritis and ganglionitis in mice: evidence for intra-axonal transport of infection.

    PubMed

    Cook, M L; Stevens, J G

    1973-02-01

    The pathogenesis of acute herpetic infection in the nervous system has been studied following rear footpad inoculation of mice. Viral assays performed on appropriate tissues at various time intervals indicated that the infection progressed sequentially from peripheral to the central nervous system, with infectious virus reaching the sacrosciatic spinal ganglia in 20 to 24 hr. The infection also progressed to ganglia in mice given high levels of anti-viral antibody. Immunofluorescent techniques demonstrated that both neurons and supporting cells produced virus-specific antigens. By electron microscopy, neurons were found to produce morphologically complete virions, but supporting cells replicated principally nucleocapsids. These results are discussed in the context of possible mechanisms by which herpes simplex virus might travel in nerve trunks. They are considered to offer strong support for centripetal transport in axons.

  15. Axonal collateral-collateral transport of tract tracers in brain neurons: false anterograde labelling and useful tool.

    PubMed

    Chen, S; Aston-Jones, G

    1998-02-01

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

  16. 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

  17. 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.

  18. 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.

  19. Axonal transport of rabies virus in the central nervous system of the rat.

    PubMed

    Gillet, J P; Derer, P; Tsiang, H

    1986-11-01

    Stereotaxic inoculation of rabies virus into specific nuclei in the central nervous system has been used for the investigation of the central neural transport mechanisms of viral information. The infection was monitored by specific fluorescence and peroxidase studies and the titration of viral infectivity in dissected brain areas. Twenty-four hours after inoculation into the striatum, cortex, or substantia nigra, infected neurons were detected only in cells from areas and nuclei which were related to the site of inoculation. The distribution of infected neurons showed that retrograde axoplasmic flow plays a determining role in the transport of rabies virus 24 hours after delivery of virus to specific target nuclei. Local destruction of neurons by kainic acid at the site of viral inoculation did not prevent the uptake and subsequent retrograde axonal transport of virus. There was an overall correlation between the major neural connections of the inoculated areas (e.g. the striatum) and the infected areas 24 hours later (e.g. the substantia nigra).

  20. 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

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

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

    Maier, C.E.

    1989-01-01

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

  3. 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.

  4. Analytical comparison between Nixon-Logvinenko's and Jung-Brown's theories of slow neurofilament transport in axons.

    PubMed

    Kuznetsov, I A; Kuznetsov, A V

    2013-10-01

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

  5. R-Flurbiprofen Improves Axonal Transport in the Tg2576 Mouse Model of Alzheimer’s Disease as Determined by MEMRI

    PubMed Central

    Smith, Karen D.B.; Paylor, Richard; Pautler, Robia G.

    2011-01-01

    Axonal pathology is a prevalent feature of Alzheimer’s disease (AD) and is thought to occur predominantly due to the accumulation of amyloid beta (Aβ). However, it remains unclear whether therapeutics geared towards reducing Aβ improves axonal deficits. We have previously used Manganese Enhanced MRI (MEMRI) to demonstrate that axonal transport deficits occur before plaque formation in the Tg2576 mouse model of AD. Here we tested whether axonal transport deficits in the Tg2576 mouse model improve in response to the Aβ42 selective lowering agent R-Flurbiprofen (R-F). We demonstrated that in young animals (before Aβ plaque formation), R-F treatment reduced Aβ42 levels and coincided with a significant improvement in axonal transport (p=0.0186) iHowever, in older animals (after plaque formation had occurred), we observed that R-F treatment did not reduce Aβ42 levels although we still observed a significant improvement in axonal transport as assessed with MEMRI (p=0.0329). We then determined that R-F treatment reduced tau hyper-phosphorylation in the older animals. These data indicate that both Aβ42 and tau comprise a role in axonal transport rate deficits in the Tg2576 models. PMID:21500269

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

    PubMed Central

    1995-01-01

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

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

    PubMed

    Hogan, E M; Cohen, M A; Boron, W F

    1995-11-01

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

  8. 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

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

    PubMed Central

    Gunter, Thomas E.; Sheu, Shey-Shing

    2009-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-24

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

  12. 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

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

    PubMed

    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

    2016-04-12

    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.

  14. 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.

  15. 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

  16. Calcium transport across the inner mitochondrial membrane: molecular mechanisms and pharmacology

    PubMed Central

    Csordás, György; Várnai, Peter; Golenár, Tünde; Sheu, Shey-Shing; Hajnóczky, György

    2011-01-01

    Growing evidence supports that mitochondrial calcium uptake is important for cell metabolism, signaling and survival. However, both the molecular nature of the mitochondrial Ca2+ transport sites and the calcium signals they respond to remained elusive. Recent RNA interference studies have identified new candidate proteins for Ca2+ uptake across the inner mitochondrial membrane, including LETM1, MCU, MICU1 and NCLX. The sensitivity of these factors to several drugs has been tested and in parallel, some new inhibitors of mitochondrial Ca2+ uptake have been described. This paper provides an update on the pharmacological aspects of the molecular mechanisms of the inner mitochondrial membrane Ca2+ transport. PMID:22123069

  17. Sec24- and ARFGAP1-dependent trafficking of GABA transporter-1 is a prerequisite for correct axonal targeting.

    PubMed

    Reiterer, Veronika; Maier, Susanne; Sitte, Harald H; Kriz, Alexander; Rüegg, Markus A; Hauri, Hans-Peter; Freissmuth, Michael; Farhan, Hesso

    2008-11-19

    The GABA transporter-1 (GAT1) is a prototypical protein of the synaptic specialization. Export of GAT1 from the endoplasmic reticulum (ER) is contingent on its interaction with the COPII (coatomer protein-II) coat subunit Sec24D. Here we show that silencing all four Sec24 isoforms strongly inhibits transport of GAT1 to the cell surface. In contrast, transport of GAT1-RL/AS, a mutant that is deficient in Sec24D recruitment, was not inhibited, suggesting a nonconventional, COPII-independent pathway. However, ARFGAP1 bound directly to the C terminus of both GAT1-RL/AS and wild-type GAT1. Surface expression of GAT1-RL/AS involved ARFGAP1. GAT1-RL/AS appeared to bypass the ER-Golgi-intermediate compartment, but its pathway to the plasma membrane still involved passage through the Golgi. Thus, the GAT1-RL/AS mutant allowed to test whether COPII-dependent ER-export is required for correct sorting of GAT1 to the axon terminal in neuronal cells. In contrast to wild-type GAT1, GAT1-RL/AS failed to be specifically enriched at the tip of neurite extensions of CAD.a cells (a neuroblastoma cell line that can be differentiated into a neuron-like phenotype) and in the axon terminals of hippocampal neurons. These findings indicate that correct sorting to the axon is contingent on ER export via the COPII machinery and passage through the ER-Golgi-intermediate compartment.

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

    PubMed Central

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

    2014-01-01

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

  19. 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.

  20. The action of puromycin and cycloheximide on the initiation of rapid axonal transport in amphibian dorsal root neurones.

    PubMed

    Nichols, T R; Smith, R S; Snyder, R E

    1982-11-01

    1. Amphibian dorsal root ganglia-sciatic nerve preparations were incubated in vitro and the rapid axonal transport of radioactive labels was studied with a position-sensitive detector and by conventional liquid scintillation analysis. Protein was labelled by exposure of the ganglia to [(35)S]methionine or [(3)H]leucine and lipid was labelled using [(32)P]orthophosphoric acid.2. Protein synthesis was interrupted by exposure of the ganglia to either cycloheximide or puromycin. When ganglia were exposed to either inhibitor prior to or simultaneously with a label, the somal export of both protein and lipid to the axon was reduced by two to three orders of magnitude.3. Using the position-sensitive detector, [(35)S]methionine was observed to be exported from the ninth dorsal root ganglia of Rana catesbiana 3.49+/-1.56 h (+/- S.D.) after exposure, and [(32)P]phosphate 4.46+/-1.85 h after exposure.4. Export of [(35)S]methionine or [(32)P]phosphate was disrupted 3.32+/-1.21 h (+/- S.D.) or 1.93+/-1.04 h respectively after exposure of the ganglia to cycloheximide or puromycin.5. For a given preparation the time required for [(35)S]methionine to be exported was statistically equal to the time required for cycloheximide or puromycin to disrupt export. No such correlation was found to exist for the export of [(32)P]phosphate.6. Analysis revealed that materials labelled with either [(35)S]methionine or [(32)P]phosphate continue to be exported from the ganglia for several hours after the initial disruption in outflow caused by the inhibitors.7. The results do not provide support for the hypothesis of Ambron, Goldman & Schwartz (1975) that a ;key' newly synthesized, and non-storable, polypeptide is added to an already assembled structure to allow rapid axonal transport to be initiated. PMID:6185671

  1. Synaptic vesicles in electromotoneurones. I. Axonal transport, site of transmitter uptake and processing of a core proteoglycan during maturation.

    PubMed Central

    Kiene, M L; Stadler, H

    1987-01-01

    We were able by using an in vivo pulse-label technique to trace part of the life cycle of a secretory organelle, the acetylcholine-storing synaptic vesicle from electromotoneurones of Torpedo marmorata. This technique uses [35S]sulphate incorporation into the cell bodies of the electromotoneurones which results in radioactive labelling of a synaptic vesicle heparansulphate proteoglycan--a major core component. Vesicles are anterogradely transported in the axons at a fast rate as 'empty' organelles (VP0 population). In the nerve terminal, maturation of the granule to a population (VP1) fully charged with acetylcholine and ATP occurs. Finally after a longer time interval a change to a third population (VP2) is observed. This population is reduced in diameter as compared to VP0 and VP1 suggesting, in agreement with earlier reports, that it has undergone exo-endocytosis. The changes from VP0 to VP1 and VP2 are accompanied by a degradation of the core proteoglycan as measured by gel filtration of the 35S-labelled compound. The results show that vesicles are axonally transported as preformed organelles, exist in the neurone at least in three different populations and that the nerve terminal is the major site of transmitter uptake. Images Fig. 1. Fig. 4. Fig. 6. PMID:2444433

  2. Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration, and premature motoneuron death and accelerates motoneuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1.

    PubMed

    Jaarsma, D; Haasdijk, E D; Grashorn, J A; Hawkins, R; van Duijn, W; Verspaget, H W; London, J; Holstege, J C

    2000-12-01

    Cytosolic Cu/Zn superoxide dismutase (SOD1) is a ubiquitous small cytosolic metalloenzyme that catalyzes the conversion of superoxide anion to hydrogen peroxide (H(2)O(2)). Mutations in the SOD1 gene cause a familial form of amyotrophic lateral sclerosis (fALS). The mechanism by which mutant SOD1s causes ALS is not understood. Transgenic mice expressing multiple copies of fALS-mutant SOD1s develop an ALS-like motoneuron disease resembling ALS. Here we report that transgenic mice expressing a high concentration of wild-type human SOD1 (hSOD1(WT)) develop an array of neurodegenerative changes consisting of (1) swelling and vacuolization of mitochondria, predominantly in axons in the spinal cord, brain stem, and subiculum; (2) axonal degeneration in a number of long fiber tracts, predominantly the spinocerebellar tracts; and (3) at 2 years of age, a moderate loss of spinal motoneurons. Parallel to the development of neurodegenerative changes, hSOD1(WT) mice also develop mild motor abnormalities. Interestingly, mitochondrial vacuolization was associated with accumulation of hSOD1 immunoreactivity, suggesting that the development of mitochondrial pathology is associated with disturbed SOD1 turnover. In this study we also crossed hSOD1(WT) mice with a line of fALS-mutant SOD1 mice (hSOD1(G93A)) to generate "double" transgenic mice that express high levels of both wild-type and G93A mutant hSOD1. The "double" transgenic mice show accelerated motoneuron death, earlier onset of paresis, and earlier death as compared with hSOD1(G93A) littermates. Thus in vivo expression of high levels of wild-type hSOD1 is not only harmful to neurons in itself, but also increases or facilitates the deleterious action of a fALS-mutant SOD1. Our data indicate that it is important for motoneurons to control the SOD1 concentration throughout their processes, and that events that lead to improper synthesis, transport, or breakdown of SOD1 causing its accumulation are potentially dangerous.

  3. 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

  4. 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

  5. Abnormal Mitochondrial L-Arginine Transport Contributes to the Pathogenesis of Heart Failure and Rexoygenation Injury

    PubMed Central

    Byrne, Melissa; Joshi, Mandar; Horlock, Duncan; Lam, Nicholas T.; Gregorevic, Paul; McGee, Sean L.; Kaye, David M.

    2014-01-01

    Background Impaired mitochondrial function is fundamental feature of heart failure (HF) and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO) metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury. Methods and Results In mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model) we demonstrated a marked reduction in L-arginine uptake (p<0.05 and p<0.01 respectively) and expression of the principal L-arginine transporter, CAT-1 (p<0.001, p<0.01) compared to controls. This was accompanied by significantly lower NO production and higher 3-nitrotyrosine levels (both p<0.05). The role of mitochondrial L-arginine transport in modulating cardiac stress responses was examined in cardiomyocytes with mitochondrial specific overexpression of CAT-1 (mtCAT1) exposed to hypoxia-reoxygenation stress. mtCAT1 cardiomyocytes had significantly improved mitochondrial membrane potential, respiration and ATP turnover together with significantly decreased reactive oxygen species production and cell death following mitochondrial stress. Conclusion These data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury. PMID:25111602

  6. 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

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

    PubMed

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

    2016-10-01

    Activity-dependent neuroprotective protein (ADNP), essential for brain formation, is a frequent autism spectrum disorder (ASD)-mutated gene. ADNP associates with microtubule end-binding proteins (EBs) through its SxIP motif, to regulate dendritic spine formation and brain plasticity. Here, we reveal SKIP, a novel four-amino-acid peptide representing an EB-binding site, as a replacement therapy in an outbred Adnp-deficient mouse model. We discovered, for the first time, axonal transport deficits in Adnp(+/-) mice (measured by manganese-enhanced magnetic resonance imaging), with significant male-female differences. RNA sequencing evaluations showed major age, sex and genotype differences. Function enrichment and focus on major gene expression changes further implicated channel/transporter function and the cytoskeleton. In particular, a significant maturation change (1 month-five months) was observed in beta1 tubulin (Tubb1) mRNA, only in Adnp(+/+) males, and sex-dependent increase in calcium channel mRNA (Cacna1e) in Adnp(+/+) males compared with females. At the protein level, the Adnp(+/-) mice exhibited impaired hippocampal expression of the calcium channel (voltage-dependent calcium channel, Cacnb1) as well as other key ASD-linked genes including the serotonin transporter (Slc6a4), and the autophagy regulator, BECN1 (Beclin1), in a sex-dependent manner. Intranasal SKIP treatment normalized social memory in 8- to 9-month-old Adnp(+/-)-treated mice to placebo-control levels, while protecting axonal transport and ameliorating changes in ASD-like gene expression. The control, all d-amino analog D-SKIP, did not mimic SKIP activity. SKIP presents a novel prototype for potential ASD drug development, a prevalent unmet medical need. PMID:26782054

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

    PubMed

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

    2016-10-01

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

  9. 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

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

    PubMed Central

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

    2016-01-01

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

  11. Rapid orthograde transport of 32P-labelled material in amphibian sensory axons: a multiwire proportional chamber study.

    PubMed

    Snyder, R E; Nichols, T R; Smith, R S

    1980-05-01

    A multiwire proportional chamber was used to follow the axonal transport of material labelled with [32P]orthophosphate in dorsal root ganglion (DRG)--sciatic nerve preparations of Xenopus laevis and Rana catesbiana. The DRG were exposed to label for a period of 4 h following which there was a period of continued delivery of labelled material to the nerve for up to 18 h. The front of the labelled material in the nerve moved at a velocity of 160--170 mm/24 h at room temperature (22.5--23.5 degrees C). Sectioning the nerve at a proximal position showed that labelled material behind the front moved at a similar rapid velocity. Experiments in which the nerve was sectioned showed that some of the rapidly transported label appeared to be deposited into a relatively stationary phase. Extrapolation of the results indicated that the delay between the presentation of the label to the DRG and the onset of the transport of labelled material in the nerve was 4--6 h. The rapid transport of the label was inhibited by vinblastine sulphate at concentrations of 130--950 microM. Most of the rapidly transported material was found to be in a chloroform-methanol extractable form. In conclusion, 32P labels materials whose transport dynamics are very similar to those observed when [35S]methionine is used as the precursor. PMID:6158368

  12. Mitochondrial fusion/fission, transport and autophagy in Parkinson's disease: when mitochondria get nasty.

    PubMed

    Arduíno, Daniela M; Esteves, A Raquel; Cardoso, Sandra M

    2011-02-20

    Understanding the molecular basis of Parkinson's disease (PD) has proven to be a major challenge in the field of neurodegenerative diseases. Although several hypotheses have been proposed to explain the molecular mechanisms underlying the pathogenesis of PD, a growing body of evidence has highlighted the role of mitochondrial dysfunction and the disruption of the mechanisms of mitochondrial dynamics in PD and other parkinsonian disorders. In this paper, we comment on the recent advances in how changes in the mitochondrial function and mitochondrial dynamics (fusion/fission, transport, and clearance) contribute to neurodegeneration, specifically focusing on PD. We also evaluate the current controversies in those issues and discuss the role of fusion/fission dynamics in the mitochondrial lifecycle and maintenance. We propose that cellular demise and neurodegeneration in PD are due to the interplay between mitochondrial dysfunction, mitochondrial trafficking disruption, and impaired autophagic clearance.

  13. The mitochondrial transporter family (SLC25): physiological and pathological implications.

    PubMed

    Palmieri, Ferdinando

    2004-02-01

    The mitochondrial carriers (MCs) shuttle a variety of metabolites across the inner mitochondrial membrane (i.m.m.). In man they are encoded by the SLC25 genes. Some MCs have isoforms encoded by different SLC25 genes, whereas the phosphate carrier has two variants arising from an alternative splicing of SLC25A3. Six MCs have been sequenced after purification, and many more have been identified from their transport and kinetic properties following heterologous over-expression and reconstitution into liposomes. All MCs of known function belong to the same protein family, since their polypeptide chains consist of three tandemly related sequences of about 100 amino acids, and the repeats of the different carriers are homologous. They probably function as homodimers, each monomer being folded in the membrane into six transmembrane segments. The functional information obtained in studies with mitochondria and/or the reconstituted system has helped to gain an insight into the physiological role of the MCs in cell metabolism, as have tissue distribution, the use of knock-out mice (and/or yeast) and over-expression in human cell lines (or yeast) of individual carriers and isoforms. At the same time, the cloning and functional identification of many SLC25 genes has made it possible (i) to identify the genes (and their defects) responsible for some diseases, e.g. Stanley syndrome and Amish microcephaly, and (ii) where the genes were already known, to characterize the function of the gene products and hence understand the molecular basis and the symptoms of the diseases, e.g. hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) syndrome and type II citrullinemia. It is likely that further extension and functional characterization of the SLC25 gene family will elucidate other diseases caused by MC deficiency. PMID:14598172

  14. Glia to axon RNA transfer.

    PubMed

    Sotelo, José Roberto; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José Roberto; Calliari, Aldo; Cal, Karina; Bresque, Mariana; Dipaolo, Andrés; Farias, Joaquina; Mercer, John A

    2014-03-01

    The existence of RNA in axons has been a matter of dispute for decades. Evidence for RNA and ribosomes has now accumulated to a point at which it is difficult to question, much of the disputes turned to the origin of these axonal RNAs. In this review, we focus on studies addressing the origin of axonal RNAs and ribosomes. The neuronal soma as the source of most axonal RNAs has been demonstrated and is indisputable. However, the surrounding glial cells may be a supplemental source of axonal RNAs, a matter scarcely investigated in the literature. Here, we review the few papers that have demonstrated that glial-to-axon RNA transfer is not only feasible, but likely. We describe this process in both invertebrate axons and vertebrate axons. Schwann cell to axon ribosomes transfer was conclusively demonstrated (Court et al. [2008]: J. Neurosci 28:11024-11029; Court et al. [2011]: Glia 59:1529-1539). However, mRNA transfer still remains to be demonstrated in a conclusive way. The intercellular transport of mRNA has interesting implications, particularly with respect to the integration of glial and axonal function. This evolving field is likely to impact our understanding of the cell biology of the axon in both normal and pathological conditions. Most importantly, if the synthesis of proteins in the axon can be controlled by interacting glia, the possibilities for clinical interventions in injury and neurodegeneration are greatly increased.

  15. 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

  16. Oxygen tolerance and coupling of mitochondrial electron transport.

    PubMed

    Campian, Jian Li; Qian, Mingwei; Gao, Xueshan; Eaton, John W

    2004-11-01

    Oxygen is critical to aerobic metabolism, but excessive oxygen (hyperoxia) causes cell injury and death. An oxygen-tolerant strain of HeLa cells, which proliferates even under 80% O2, termed "HeLa-80," was derived from wild-type HeLa cells ("HeLa-20") by selection for resistance to stepwise increases of oxygen partial pressure. Surprisingly, antioxidant defenses and susceptibility to oxidant-mediated killing do not differ between these two strains of HeLa cells. However, under both 20 and 80% O2, intracellular reactive oxygen species (ROS) production is significantly (approximately 2-fold) less in HeLa-80 cells. In both cell lines the source of ROS is evidently mitochondrial. Although HeLa-80 cells consume oxygen at the same rate as HeLa-20 cells, they consume less glucose and produce less lactic acid. Most importantly, the oxygen-tolerant HeLa-80 cells have significantly higher cytochrome c oxidase activity (approximately 2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. Indeed, preferential inhibition of cytochrome c oxidase by treatment with n-methyl protoporphyrin (which selectively diminishes synthesis of heme a in cytochrome c oxidase) enhances ROS production and abrogates the oxygen tolerance of the HeLa-80 cells. Thus, it appears that the remarkable oxygen tolerance of these cells derives from tighter coupling of the electron transport chain. PMID:15328348

  17. 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

  18. Facilitated mitochondrial import of antiviral and anticancer nucleoside drugs by human equilibrative nucleoside transporter-3

    PubMed Central

    Govindarajan, Rajgopal; Leung, George P. H.; Zhou, Mingyan; Tse, Chung-Ming; Wang, Joanne; Unadkat, Jashvant D

    2009-01-01

    human equilibrative nucleoside transporter-3 (hENT3) was recently reported as a pH-dependent, intracellular (lysosomal) transporter capable of transporting anti-human immunodeficiency virus (HIV) dideoxynucleosides (ddNs). Because most anti-HIV ddNs (e.g., zidovudine, AZT) exhibit clinical mitochondrial toxicity, we investigated whether hENT3 facilitates transport of anti-HIV ddNs into the mitochondria. Cellular fractionation and immunofluorescence microscopy studies in several human cell lines identified a substantial presence of hENT3 in the mitochondria, with additional presence at the cell surface of two placental cell lines (JAR, JEG3). Mitochondrial or cell surface hENT3 expression was confirmed in human hepatocytes and placental tissues, respectively. Unlike endogenous hENT3, yellow fluorescent protein (YFP)-tagged hENT3 was partially directed to the lysosomes. Xenopus oocytes expressing NH2-terminal-deleted hENT3 (expressed at the cell surface) showed pH-dependent interaction with several classes of nucleosides (anti-HIV ddNs, gemcitabine, fialuridine, ribavirin) that produce mitochondrial toxicity. Transport studies in hENT3 gene-silenced JAR cells showed significant reduction in mitochondrial transport of nucleosides and nucleoside drugs. Our data suggest that cellular localization of hENT3 is cell type dependent and the native transporter is substantially expressed in mitochondria and/or cell surface. hENT3-mediated mitochondrial transport may play an important role in mediating clinically observed mitochondrial toxicity of nucleoside drugs. In addition, our finding that hENT3 is a mitochondrial transporter is consistent with the recent finding that mutations in the hENT3 gene cause an autosomal recessive disorder in humans called the H syndrome. PMID:19164483

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Function and regulation of local axonal translation

    PubMed Central

    Lin, Andrew C; Holt, Christine E

    2013-01-01

    An increasing body of evidence indicates that local axonal translation is required for growing axons to respond appropriately to guidance cues and other stimuli. Recent studies suggest that asymmetrical synthesis of cytoskeletal proteins mediates growth cone turning and that local translation and retrograde transport of transcription factors mediate neuronal survival. Axonal translation is regulated partly by selective axonal localization of mRNAs and by translation initiation factors and RNA-binding proteins. We discuss possible rationales for local axonal translation, including distinct properties of nascent proteins, precise localization, and axonal autonomy. PMID:18508259

  4. Mitochondrial transporters of the SLC25 family and associated diseases: a review.

    PubMed

    Palmieri, Ferdinando

    2014-07-01

    To date, 14 inherited diseases (including phenotypes) associated to mitochondrial transporters of the SLC25 family have been well characterized biochemically and genetically. They are rare metabolic disorders caused by mutations in the SLC25 nuclear genes that encode mitochondrial carriers, a superfamily of 53 proteins in humans that shuttle a variety of solutes across the mitochondrial membrane. Mitochondrial carriers vary considerably in the nature and size of the substrates they transport, the modes of transport and driving forces. However, their substrate translocation mechanism at the molecular level is thought to be basically the same. Herein, the main structural and functional properties of the SLC25 mitochondrial carriers and the known carrier-related diseases are presented. Two of these disorders, ADP/ATP carrier deficiency and phosphate carrier deficiency, are caused by defects of the two mitochondrial carriers that provide mitochondria with ADP and phosphate, the substrates of oxidative phosphorylation; these disorders therefore are characterized by defective energy production by mitochondria. The mutations of SLC25 carrier genes involved in other cellular functions cause carnitine/acylcarnitine carrier deficiency, HHH syndrome, aspartate/glutamate isoform 1 and 2 deficiencies, congenital Amish microcephaly, neuropathy with bilateral striatal necrosis, congenital sideroblastic anemia, neonatal epileptic encephalopathy, and citrate carrier deficiency; these disorders are characterized by specific metabolic dysfunctions depending on the role of the defective carrier in intermediary metabolism. PMID:24797559

  5. [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

  6. Coordinating Gene Expression and Axon Assembly to Control Axon Growth: Potential Role of GSK3 Signaling

    PubMed Central

    Liu, Chang-Mei; Hur, Eun-Mi; Zhou, Feng-Quan

    2012-01-01

    Axon growth requires the coordinated regulation of gene expression in the neuronal soma, local protein translation in the axon, anterograde transport of synthesized raw materials along the axon, and assembly of cytoskeleton and membranes in the nerve growth cone. Glycogen synthase kinase 3 (GSK3) signaling has recently been shown to play key roles in the regulation of axonal transport and cytoskeletal assembly during axon growth. GSK3 signaling is also known to regulate gene expression via controlling the functions of many transcription factors, suggesting that GSK3 may be an important regulator of gene transcription supporting axon growth. We review signaling pathways that control local axon assembly at the growth cone and gene expression in the soma during developmental or regenerative axon growth and discuss the potential involvement of GSK3 signaling in these processes, with a particular focus on how GSK3 signaling modulates the function of axon growth-associated transcription factors. PMID:22347166

  7. Pressure-induced inhibition of fast axonal transport of proteins in the rabbit vagus nerve in galactose neuropathy: prevention by an aldose reductase inhibitor.

    PubMed

    McLean, W G

    1988-07-01

    Fast and slow anterograde axonal transport and retrograde axonal transport of proteins were studied in the mainly non-myelinated sensory fibres of the vagus nerve of rabbits fed a diet of 50% galactose over a period of 29 days. Galactose feeding had no effect on the rate or protein composition of slow transport nor on the amount of retrogradely transported proteins. There was a slight retardation of fast transported proteins although their composition was unchanged. The galactose feeding led to a significant increase (p less than 0.005) in nerve water content and nerve galactitol but no significant change in myo-inositol. When 20 mm Hg pressure was applied locally to the cervical vagus nerve, fast transported proteins accumulated proximal to the compression zone in the galactose-fed but not in control rabbits. Administration of the aldose reductase inhibitor Statil (ICI 128436) throughout the experiment prevented the increased susceptibility to pressure and the increase in nerve galactitol and water content. The effects of pressure are similar to those found in the streptozotocin-diabetic rat although the underlying mechanisms may differ.

  8. Pyruvate fuels mitochondrial respiration and proliferation of breast cancer cells: effect of monocarboxylate transporter inhibition.

    PubMed

    Diers, Anne R; Broniowska, Katarzyna A; Chang, Ching-Fang; Hogg, Neil

    2012-06-15

    Recent studies have highlighted the fact that cancer cells have an altered metabolic phenotype, and this metabolic reprogramming is required to drive the biosynthesis pathways necessary for rapid replication and proliferation. Specifically, the importance of citric acid cycle-generated intermediates in the regulation of cancer cell proliferation has been recently appreciated. One function of MCTs (monocarboxylate transporters) is to transport the citric acid cycle substrate pyruvate across the plasma membrane and into mitochondria, and inhibition of MCTs has been proposed as a therapeutic strategy to target metabolic pathways in cancer. In the present paper, we examined the effect of different metabolic substrates (glucose and pyruvate) on mitochondrial function and proliferation in breast cancer cells. We demonstrated that cancer cells proliferate more rapidly in the presence of exogenous pyruvate when compared with lactate. Pyruvate supplementation fuelled mitochondrial oxygen consumption and the reserve respiratory capacity, and this increase in mitochondrial function correlated with proliferative potential. In addition, inhibition of cellular pyruvate uptake using the MCT inhibitor α-cyano-4-hydroxycinnamic acid impaired mitochondrial respiration and decreased cell growth. These data demonstrate the importance of mitochondrial metabolism in proliferative responses and highlight a novel mechanism of action for MCT inhibitors through suppression of pyruvate-fuelled mitochondrial respiration.

  9. Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production.

    PubMed

    Ye, Junli; Jiang, Zhongxin; Chen, Xuehong; Liu, Mengyang; Li, Jing; Liu, Na

    2016-01-15

    Reactive oxygen species (ROS) are believed to be mediators of excessive microglial activation, yet the resources and mechanism are not fully understood. Here we stimulated murine microglial BV-2 cells and primary microglial cells with different inhibitors of electron transport chain (ETC), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, and NaN3 to induce mitochondrial ROS production and we observed the role of mitochondrial ROS in microglial activation. Our results showed that ETC inhibitors resulted in significant changes in cell viability, microglial morphology, cell cycle arrest and mitochondrial ROS production in a dose-dependent manner in both primary cultural microglia and BV-2 cell lines. Moreover, ETC inhibitors, especially rotenone and antimycin A stimulated secretion of interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) by microglia with marked activation of mitogen-activated proteinkinases (MAPKs) and nuclear factor κB (NF-κB), which could be blocked by specific inhibitors of MAPK and NF-κB and mitochondrial antioxidants, Mito-TEMPO. Taken together, our results demonstrated that inhibition of mitochondrial respiratory chain in microglia led to production of mitochondrial ROS and therefore may activate MAPK/NF-кB dependent inflammatory cytokines release in microglia, which indicated that mitochondrial-derived ROS were contributed to microglial activation.

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

    PubMed

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

    2000-08-01

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

  11. KIF1A, an Axonal Transporter of Synaptic Vesicles, Is Mutated in Hereditary Sensory and Autonomic Neuropathy Type 2

    PubMed Central

    Rivière, Jean-Baptiste; Ramalingam, Siriram; Lavastre, Valérie; Shekarabi, Masoud; Holbert, Sébastien; Lafontaine, Julie; Srour, Myriam; Merner, Nancy; Rochefort, Daniel; Hince, Pascale; Gaudet, Rébecca; Mes-Masson, Anne-Marie; Baets, Jonathan; Houlden, Henry; Brais, Bernard; Nicholson, Garth A.; Van Esch, Hilde; Nafissi, Shahriar; De Jonghe, Peter; Reilly, Mary M.; Timmerman, Vincent; Dion, Patrick A.; Rouleau, Guy A.

    2011-01-01

    Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system. PMID:21820098

  12. An ABC transporter in the mitochondrial inner membrane is required for normal growth of yeast.

    PubMed Central

    Leighton, J; Schatz, G

    1995-01-01

    In an attempt to identify a mitochondrial ATP binding cassette (ABC) transporter, we have used the polymerase chain reaction to amplify 10 DNA fragments homologous to members of the ABC family from the yeast Saccharomyces cerevisiae. We disrupted five of the corresponding genes and found that one of the resulting null mutants barely grew on rich medium and failed to grow on minimal medium. This gene, termed ATM1, encodes a putative 'half-transporter' of 694 amino acids. Atm1p is synthesized with an N-terminal mitochondrial matrix-targeting signal and is located in the mitochondrial inner membrane, with its C-terminal ATPase domain exposed to the matrix. Cells lacking a functional ATM1 gene have an unstable mitochondrial genome and have white mitochondria that completely lack cytochromes. Atm1p is the first mitochondrial member of the ABC family to be identified and the only eukaryotic ABC transporter that has been shown to be necessary for normal cellular growth. Images PMID:7828591

  13. 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.

  14. KIF5B promotes the forward transport and axonal function of the voltage-gated sodium channel Nav1.8.

    PubMed

    Su, Yuan-Yuan; Ye, Mingyu; Li, Lei; Liu, Chao; Pan, Jing; Liu, Wen-Wen; Jiang, Yanbo; Jiang, Xing-Yu; Zhang, Xu; Shu, Yousheng; Bao, Lan

    2013-11-01

    Nav1.8 is a tetrodotoxin-resistant voltage-gated sodium channel selectively expressed in primary sensory neurons. Peripheral inflammation and nerve injury induce Nav1.8 accumulation in peripheral nerves. However, the mechanisms and related significance of channel accumulation in nerves remains unclear. Here we report that KIF5B promotes the forward transport of Nav1.8 to the plasma membrane and axons in dorsal root ganglion (DRG) neurons of the rat. In peripheral inflammation induced through the intraplantar injection of complete Freund's adjuvant, increased KIF5 and Nav1.8 accumulation were observed in the sciatic nerve. The knock-down of KIF5B, a highly expressed member of the KIF5 family in DRGs, reduced the current density of Nav1.8 in both cultured DRG neurons and ND7-23 cells. Overexpression of KIF5B in ND7-23 cells increased the current density and surface expression of Nav1.8, which were abolished through brefeldin A treatment, whereas the increases were lost in KIF5B mutants defective in ATP hydrolysis or cargo binding. Overexpression of KIF5B also decreased the proteasome-associated degradation of Nav1.8. In addition, coimmunoprecipitation experiments showed interactions between the N terminus of Nav1.8 and the 511-620 aa sequence in the stalk domain of KIF5B. Furthermore, KIF5B increased Nav1.8 accumulation, Nav1.8 current, and neuronal excitability detected in the axons of cultured DRG neurons, which were completely abolished by the disruption of interactions between KIF5B and the N terminus of Nav1.8. Therefore, our results reveal that KIF5B is required for the forward transport and axonal function of Nav1.8, suggesting a mechanism for axonal accumulation of Nav1.8 in inflammatory pain. PMID:24198377

  15. Insights into the mutation-induced HHH syndrome from modeling human mitochondrial ornithine transporter-1.

    PubMed

    Wang, Jing-Fang; Chou, Kuo-Chen

    2012-01-01

    Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109-113). Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i) the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii) the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii) the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and developing effective

  16. 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

  17. 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

  18. 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

  19. 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

  20. Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana.

    PubMed

    Lee, Chun Pong; Wirtz, Markus; Hell, Rüdiger

    2014-01-01

    Cysteine is essential for many mitochondrial processes in plants, including translation, iron-sulfur cluster biogenesis and cyanide detoxification. Its biosynthesis is carried out by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which can be found in the cytosol, plastids and mitochondria. Mutants lacking one compartment-specific OAS-TL isoform show viable phenotypes, leading to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway. In this report, we show that exogenouslly supplied [(35)S]cysteine accumulates in the mitochondrial fraction and is taken up into isolated mitochondria for in organello protein synthesis. Analysis of cysteine uptake by isolated mitochondria and mitoplasts indicates that cysteine is transported by multiple facilitated mechanisms that operate in a concentration gradient-dependent manner. In addition, cysteine uptake is dependent mainly on the ΔpH across the inner membrane. The rates of mitochondrial cysteine transport can be mildly altered by specific metabolites in the cyanide detoxification-linked sulfide oxidation, but not by most substrates and products of the cysteine biosynthetic pathway. Based on these results, we propose that the transport of cysteine plays a pivotal role in regulating cellular cysteine biosynthesis as well as modulating the availability of sulfur for mitochondrial metabolism.

  1. 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

  2. Axonal change in minor head injury.

    PubMed

    Povlishock, J T; Becker, D P; Cheng, C L; Vaughan, G W

    1983-05-01

    Anterograde axonal transport of horseradish peroxidase (HRP) in selected cerebral and cerebellar efferents was studied in cats subjected to minor head injury. After trauma, the animals were allowed to survive from one to 24 hours, when they were perfused with aldehydes and processed for the light and electron microscopic visualization of the peroxidase reaction product. By light microscopy, the brain injury elicited an initial intra-axonal peroxidase pooling. With longer post-traumatic survival, HRP pooling increased in size, demonstrated frequent lobulation, and ultimately formed large ball- or club-like swellings which suggested frank axonal separation from the distal axonal segment. Ultrastructural examination revealed that the initial intra-axonal peroxidase pooling was associated with organelle accumulation which occurred without any other form of axonal change or related parenchymal or vascular damage. This accumulation of organelles increased with time and was associated with conspicuous axonal swelling. Ultimately these organelle-laden swellings lost continuity with the distal axonal segment and the axonal swelling was either completely invested by a thin myelin sheath or protruded without myelin investment into the brain parenchyma. This study suggests that axonal change is a consistent feature of minor head injury. Since these axonal changes occurred without any evidence of focal parenchymal or vascular damage, minor brain injury may ultimately disrupt axons without physically shearing or tearing them. PMID:6188807

  3. High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system.

    PubMed

    Hall, Arnaldur; Larsen, Anna K; Parhamifar, Ladan; Meyle, Kathrine D; Wu, Lin-Ping; Moghimi, S Moein

    2013-10-01

    Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems. PMID:23850549

  4. 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

  5. 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

  6. 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.

  7. Arabidopsis PPR40 Connects Abiotic Stress Responses to Mitochondrial Electron Transport1[W][OA

    PubMed Central

    Zsigmond, Laura; Rigó, Gábor; Szarka, András; Székely, Gyöngyi; Ötvös, Krisztina; Darula, Zsuzsanna; Medzihradszky, Katalin F.; Koncz, Csaba; Koncz, Zsuzsa; Szabados, László

    2008-01-01

    Oxidative respiration produces adenosine triphosphate through the mitochondrial electron transport system controlling the energy supply of plant cells. Here we describe a mitochondrial pentatricopeptide repeat (PPR) domain protein, PPR40, which provides a signaling link between mitochondrial electron transport and regulation of stress and hormonal responses in Arabidopsis (Arabidopsis thaliana). Insertion mutations inactivating PPR40 result in semidwarf growth habit and enhanced sensitivity to salt, abscisic acid, and oxidative stress. Genetic complementation by overexpression of PPR40 complementary DNA restores the ppr40 mutant phenotype to wild type. The PPR40 protein is localized in the mitochondria and found in association with Complex III of the electron transport system. In the ppr40-1 mutant the electron transport through Complex III is strongly reduced, whereas Complex IV is functional, indicating that PPR40 is important for the ubiqinol-cytochrome c oxidoreductase activity of Complex III. Enhanced stress sensitivity of the ppr40-1 mutant is accompanied by accumulation of reactive oxygen species, enhanced lipid peroxidation, higher superoxide dismutase activity, and altered activation of several stress-responsive genes including the alternative oxidase AOX1d. These results suggest a close link between regulation of oxidative respiration and environmental adaptation in Arabidopsis. PMID:18305213

  8. Isolation and Analyses of Axonal Ribonucleoprotein Complexes

    PubMed Central

    Doron-Mandel, Ella; Alber, Stefanie; Oses, Juan A.; Medzihradszky, Katalin F.; Burlingame, Alma L.; Fainzilber, Mike; Twiss, Jeffery L.; Lee, Seung Joon

    2016-01-01

    Cytoskeleton-dependent RNA transport and local translation in axons are gaining increased attention as key processes in the maintenance and functioning of neurons. Specific axonal transcripts have been found to play roles in many aspects of axonal physiology including axon guidance, axon survival, axon to soma communication, injury response and regeneration. This axonal transcriptome requires long-range transport that is achieved by motor proteins carrying transcripts as messenger ribonucleoprotein (mRNP) complexes along microtubules. Other than transport, the mRNP complex plays a major role in the generation, maintenance and regulation of the axonal transcriptome. Identification of axonal RNA binding proteins (RBPs) and analyses of the dynamics of their mRNPs are of high interest to the field. Here we describe methods for the study of interactions between RNA and proteins in axons. First, we describe a protocol for identifying binding proteins for an RNA of interest by using RNA affinity chromatography. Subsequently, we discuss immunoprecipitation (IP) methods allowing the dissection of protein- RNA and protein-protein interactions in mRNPs under various physiological conditions. PMID:26794529

  9. 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.

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

    PubMed Central

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

    2015-01-01

    The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16’s organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-α). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(−) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADα (a SAD-1-interacting protein) in the CSS system. In an unc-16(−) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(−) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16’s organelle transport regulatory function. PMID:26354976

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

    PubMed

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

    2015-09-01

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

  12. 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.

  13. Preserve and protect: maintaining axons within functional circuits.

    PubMed

    Pease, Sarah E; Segal, Rosalind A

    2014-10-01

    During development, neural circuits are initially generated by exuberant innervation and are rapidly refined by selective preservation and elimination of axons. The establishment and maintenance of functional circuits therefore requires coordination of axon survival and degeneration pathways. Both developing and mature circuits rely on interdependent mitochondrial and cytoskeletal components to maintain axonal health and homeostasis; injury or diseases that impinge on these components frequently cause pathologic axon loss. Here, we review recent findings that identify mechanisms of axonal preservation in the contexts of development, injury, and disease. PMID:25167775

  14. 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

  15. 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

  16. 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 ...

  17. 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

  18. 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.

  19. HDAC1 nuclear export induced by pathological conditions is essential for the onset of axonal damage

    PubMed Central

    Kim, Jin Young; Shen, Siming; Dietz, Karen; He, Ye; Howell, Owain; Reynolds, Richard; Casaccia, Patrizia

    2010-01-01

    Histone deacetylase 1 (HDAC1) is a nuclear enzyme involved in transcriptional repression. We report here that cytosolic HDAC1 is detected in damaged axons in brains of human patients with Multiple Sclerosis and of mice with cuprizone-induced demyelination, ex vivo models of demyelination and in cultured neurons exposed to glutamate and TNF-α. Nuclear export of HDAC1 is mediated by the interaction with the nuclear receptor CRM-1 and leads to impaired mitochondrial transport. The formation of complexes between exported HDAC1 and members of the kinesin family of motor proteins hinders the interaction with cargo molecules thereby inhibiting mitochondrial movement and inducing localized beadings. This effect is prevented by inhibiting HDAC1 nuclear export with leptomycin B, treating neurons with pharmacological inhibitors of HDAC activity or silencing HDAC1 but not other HDAC isoforms. Together these data identify nuclear export of HDAC1 as a critical event for impaired mitochondrial transport in damaged neurons. PMID:20037577

  20. Control and Regulation of Integrated Mitochondrial Function in Metabolic and Transport Networks

    PubMed Central

    Cortassa, Sonia; O’Rourke, Brian; Winslow, Raimond L.; Aon, Miguel A.

    2009-01-01

    The pattern of flux and concentration control coefficients in an integrated mitochondrial energetics model is examined by applying a generalized matrix method of control analysis to calculate control coefficients, as well as response coefficients The computational model of Cortassa et al. encompasses oxidative phosphorylation, the TCA cycle, and Ca2+ dynamics. Control of ATP synthesis, TCA cycle, and ANT fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP/ADP production and transport, as well as by Ca2+ dynamics. The calculation also analyzed the control of the concentrations of key regulatory ions and metabolites (Ca2+, NADH, ADP). The approach we have used demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis. PMID:19468321

  1. 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.

  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. 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

  4. 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

  5. Axonal oscillations in developing mammalian nerve axons

    NASA Astrophysics Data System (ADS)

    Zeng, Shangyou; Jung, Peter

    2005-01-01

    We study neuronal spike propagation in a developing myelinated axon in various stages of its development through detailed computational modeling. Recently, a form of bursting (axonal bursting), has been reported in axons in developing nerves in the absence of potassium channels. We present a computational study using a detailed model for a myelinated nerve in development to explore under what circumstances such an effect can be expected. It is shown that axonal oscillation may be caused by backfiring between the nodes of Ranvier or through backfiring from internodal sodium channels or by reducing the thickness of the myelin wrapping the axon between the nodes of Ranvier.

  6. Diabetes induces changes in KIF1A, KIF5B and dynein distribution in the rat retina: implications for axonal transport.

    PubMed

    Baptista, Filipa I; Pinto, Maria J; Elvas, Filipe; Martins, Tiago; Almeida, Ramiro D; Ambrósio, António F

    2014-10-01

    Diabetic retinopathy is a leading cause of vision loss and blindness. Disruption of axonal transport is associated with many neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous system. We investigated the impact of type 1 diabetes (2 and 8 weeks duration) on KIF1A, KIF5B and dynein motor proteins in the retina. Additionally, since hyperglycemia is considered the main trigger of diabetic complications, we investigated whether prolonged exposure to elevated glucose could affect the content and distribution of motor proteins in retinal cultures. The immunoreactivity of motor proteins was evaluated by immunohistochemistry in retinal sections and by immunoblotting in total retinal extracts from streptozotocin-induced diabetic and age-matched control animals. Primary retinal cultures were exposed to high glucose (30 mM) or mannitol (osmotic control; 24.5 mM plus 5.5 mM glucose), for seven days. Diabetes decreased the content of KIF1A at 8 weeks of diabetes as well as KIF1A immunoreactivity in the majority of retinal layers, except for the photoreceptor and outer nuclear layer. Changes in KIF5B immunoreactivity were also detected by immunohistochemistry in the retina at 8 weeks of diabetes, being increased at the photoreceptor and outer nuclear layer, and decreased in the ganglion cell layer. Regarding dynein immunoreactivity there was an increase in the ganglion cell layer after 8 weeks of diabetes. No changes were detected in retinal cultures. These alterations suggest that axonal transport may be impaired under diabetes, which might contribute to early signs of neural dysfunction in the retina of diabetic patients and animal models. PMID:25064602

  7. Neuronal Activity and Glutamate Uptake Decrease Mitochondrial Mobility in Astrocytes and Position Mitochondria Near Glutamate Transporters

    PubMed Central

    Jackson, Joshua G.; O'Donnell, John C.; Takano, Hajime; Coulter, Douglas A.

    2014-01-01

    Within neurons, mitochondria are nonuniformly distributed and are retained at sites of high activity and metabolic demand. Glutamate transport and the concomitant activation of the Na+/K+-ATPase represent a substantial energetic demand on astrocytes. We hypothesized that mitochondrial mobility within astrocytic processes might be regulated by neuronal activity and glutamate transport. We imaged organotypic hippocampal slice cultures of rat, in which astrocytes maintain their highly branched morphologies and express glutamate transporters. Using time-lapse confocal microscopy, the mobility of mitochondria within individual astrocytic processes and neuronal dendrites was tracked. Within neurons, a greater percentage of mitochondria were mobile than in astrocytes. Furthermore, they moved faster and farther than in astrocytes. Inhibiting neuronal activity with tetrodotoxin (TTX) increased the percentage of mobile mitochondria in astrocytes. Mitochondrial movement in astrocytes was inhibited by vinblastine and cytochalasin D, demonstrating that this mobility depends on both the microtubule and actin cytoskeletons. Inhibition of glutamate transport tripled the percentage of mobile mitochondria in astrocytes. Conversely, application of the transporter substrate d-aspartate reversed the TTX-induced increase in the percentage of mobile mitochondria. Inhibition of reversed Na+/Ca2+ exchange also increased the percentage of mitochondria that were mobile. Last, we demonstrated that neuronal activity increases the probability that mitochondria appose GLT-1 particles within astrocyte processes, without changing the proximity of GLT-1 particles to VGLUT1. These results imply that neuronal activity and the resulting clearance of glutamate by astrocytes regulate the movement of astrocytic mitochondria and suggest a mechanism by which glutamate transporters might retain mitochondria at sites of glutamate uptake. PMID:24478345

  8. 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

  9. 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

  10. Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis

    PubMed Central

    Cho, J; Seo, J; Lim, C H; Yang, L; Shiratsuchi, T; Lee, M-H; Chowdhury, R R; Kasahara, H; Kim, J-S; Oh, S P; Lee, Y J; Terada, N

    2015-01-01

    Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress. PMID:25613378

  11. Age-associated decline in mitochondrial respiration and electron transport in Drosophila melanogaster

    PubMed Central

    2005-01-01

    The principal objective of the present study was to identify specific alterations in mitochondrial respiratory functions during the aging process. Respiration rates and the activities of electron transport chain complexes were measured at various ages in mitochondria isolated from thoraces of the fruit fly, Drosophila melanogaster, which consist primarily of flight muscles. The rates of state 3 respiration (ADP-stimulated), RCRs (respiratory control ratios) and uncoupled respiration rates decreased significantly as a function of age, using either NAD+- or FAD-linked substrates; however, there were no differences in state 4 respiration (ADP-depleted) rates. There was also a significant age-related decline in the activity of cytochrome c oxidase (complex IV), but not of the other mitochondrial oxidoreductases examined. Exposure of mitochondria isolated from young flies to low doses of KCN or NaAz (sodium azide), complex IV inhibitors, decreased cytochrome c oxidase activity and increased the production of H2O2. Collectively, these results support the hypothesis that impairment of mitochondrial respiration may be a causal factor in the aging process, and that such impairment may result from and contribute to increased H2O2 production in vivo. PMID:15853766

  12. Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis.

    PubMed

    Cho, J; Seo, J; Lim, C H; Yang, L; Shiratsuchi, T; Lee, M-H; Chowdhury, R R; Kasahara, H; Kim, J-S; Oh, S P; Lee, Y J; Terada, N

    2015-09-01

    Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress. PMID:25613378

  13. Axonal Amphoterin mRNA Is Regulated by Translational Control and Enhances Axon Outgrowth

    PubMed Central

    Merianda, Tanuja T.; Coleman, Jennifer; Kim, Hak Hee; Kumar Sahoo, Pabitra; Gomes, Cynthia; Brito-Vargas, Paul; Rauvala, Heikki; Blesch, Armin; Yoo, Soonmoon

    2015-01-01

    High mobility group (HMG) proteins concentrate in the nucleus, interacting with chromatin. Amphoterin is an HMG protein (HMGB1) that has been shown to have extranuclear functions and can be secreted from some cell types. Exogenous amphoterin can increase neurite growth, suggesting that the secreted protein may have growth promoting activities in neurons. Consistent with this, we show that depletion of amphoterin mRNA from cultured adult rat DRG neurons attenuates neurite outgrowth, pointing to autocrine or paracrine mechanisms for its growth-promoting effects. The mRNA encoding amphoterin localizes to axonal processes and we showed recently that its 3′-UTR is sufficient for axonal localization of heterologous transcripts (Donnelly et al., 2013). Here, we show that amphoterin mRNA is transported constitutively into axons of adult DRG neurons. A preconditioning nerve injury increases the levels of amphoterin protein in axons without a corresponding increase in amphoterin mRNA in the axons. A 60 nucleotide region of the amphoterin mRNA 3′-UTR is necessary and sufficient for its localization into axons of cultured sensory neurons. Amphoterin mRNA 3′-UTR is also sufficient for axonal localization in distal axons of DRG neurons in vivo. Overexpression of axonally targeted amphoterin mRNA increases axon outgrowth in cultured sensory neurons, but axon growth is not affected when the overexpressed mRNA is restricted to the cell body. PMID:25855182

  14. A mitochondrial monocarboxylate transporter in rat liver and heart and its possible function in cell control.

    PubMed Central

    Mowbray, J

    1975-01-01

    Several hydroxy- and keto-substituted monocarboxylates were found to undergo co- as well as counter-exchange across the mitochondrial membrane. The results argue against a simple Donnan system and may be explained by the existence of a transporter for monocarboxylates. In support of this explanation it was apparently possible to 'pump' pyruvate to the sucrose-inaccessible space by using the dicarboxylate transporter. Further, several aromatic and aliphatic analogues of pyruvate, but not of di- or tri-carboxylate transport inhibitors, have been shown to prevent pyruvate-exchange reactions. Palmitoylcarnitine was found to have a much stronger affinity for the carrier than either carnitine or pyruvate and the possible consequences of this for carnitine-palmitoylcarnitine exchange and on the control of the pyruvate dehydrogenase complex are explored. In view of the range of transport inhibitors and substrates it is suggested that the carrier has a fairly broad specificity. 'Inhibitor-stop' kinetic studies show that the speed of translocation of pyruvate at 1 degrees C is of the same order as malate. The possible correlation between the role of a hydroxy-keto acid transporter in substrate exchange and some whole animal experiments is briefly discussed. It is proposed that for reasons of control the cell will require membrane monocarboxylate transporters no less than di- or tri-carboxylate carriers. PMID:1156399

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

    PubMed

    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.

  16. 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

  17. Mitochondrial transporters involved in oleic acid utilization and glutamate metabolism in yeast.

    PubMed

    Trotter, Pamela J; Adamson, Amy L; Ghrist, Angela C; Rowe, Lindsay; Scott, Lori R; Sherman, Matthew P; Stites, Nicole C; Sun, Yue; Tawiah-Boateng, Mary Anne; Tibbetts, Anne S; Wadington, Megan C; West, Aaron C

    2005-10-01

    Utilization of fatty acids such as oleic acid as sole carbon source by the yeast Saccharomyces cerevisiae requires coordinated function of peroxisomes, where the fatty acids are degraded, and the mitochondria, where oxidation is completed. We identified two mitochondrial oxodicarboxylate transporters, Odc1p and Odc2p, as important in efficient utilization of oleic acid in yeast [Tibbetts et al., Arch. Biochem. Biophys. 406 (2002) 96-104]. Yet, the growth phenotype of odc1delta odc2delta strains indicated that additional transporter(s) were also involved. Here, we identify two putative transporter genes, YMC1 and YMC2, as able to suppress the odc1delta odc2delta growth phenotype. The mRNA levels for both are elevated in the presence of glycerol or oleic acid, as compared to glucose. Ymc1p and Ymc2p are localized to the mitochondria in oleic acid-grown cells. Deletion of all four transporters (quad mutant) prevents growth on oleic acid as sole carbon source, while growth on acetate is retained. It is known that the glutamate-sensitive retrograde signaling pathway is important for upregulation of peroxisomal function in response to oleic acid and the oxodicarboxylate alpha-ketoglutarate is transported out of the mitochondria for synthesis of glutamate. So, citric acid cycle function and glutamate synthesis were examined in transporter mutants. The quad mutant has significantly decreased citrate synthase activity and whole cell alpha-ketoglutarate levels, while isocitrate dehydrogenase activity is unaffected and glutamate dehydrogenase activity is increased 10-fold. Strains carrying only two or three transporter deletions exhibit intermediate affects. 13C NMR metabolic enrichment experiments confirm a defect in glutamate biosynthesis in the quad mutant and, in double and triple mutants, suggest increased cycling of the glutamate backbone in the mitochondria before export. Taken together these studies indicate that these four transporters have overlapping activity, and

  18. 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.

  19. Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2.

    PubMed

    Muñoz-Montesino, Carola; Roa, Francisco J; Peña, Eduardo; González, Mauricio; Sotomayor, Kirsty; Inostroza, Eveling; Muñoz, Carolina A; González, Iván; Maldonado, Mafalda; Soliz, Carlos; Reyes, Alejandro M; Vera, Juan Carlos; Rivas, Coralia I

    2014-05-01

    Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport Km of 0.6mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is

  20. Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae.

    PubMed

    Cavero, S; Vozza, A; del Arco, A; Palmieri, L; Villa, A; Blanco, E; Runswick, M J; Walker, J E; Cerdán, S; Palmieri, F; Satrústegui, J

    2003-11-01

    The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae. On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast. PMID:14622413

  1. 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

  2. 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 ...

  3. 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

  4. The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

    PubMed

    Aluvila, Sreevidya; Kotaria, Rusudan; Sun, Jiakang; Mayor, June A; Walters, D Eric; Harrison, David H T; Kaplan, Ronald S

    2010-08-27

    The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

  5. 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

  6. Evolutionary Origin of the Mitochondrial Cholesterol Transport Machinery Reveals a Universal Mechanism of Steroid Hormone Biosynthesis in Animals

    PubMed Central

    Fan, Jinjiang; Papadopoulos, Vassilios

    2013-01-01

    Steroidogenesis begins with the transport of cholesterol from intracellular stores into mitochondria via a series of protein-protein interactions involving cytosolic and mitochondrial proteins located at both the outer and inner mitochondrial membranes. In adrenal glands and gonads, this process is accelerated by hormones, leading to the production of high levels of steroids that control tissue development and function. A hormone-induced multiprotein complex, the transduceosome, was recently identified, and is composed of cytosolic and outer mitochondrial membrane proteins that control the rate of cholesterol entry into the outer mitochondrial membrane. More recent studies unveiled the steroidogenic metabolon, a bioactive, multimeric protein complex that spans the outer-inner mitochondrial membranes and is responsible for hormone-induced import, segregation, targeting, and metabolism of cholesterol by cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1) in the inner mitochondrial membrane. The availability of genome information allowed us to systematically explore the evolutionary origin of the proteins involved in the mitochondrial cholesterol transport machinery (transduceosome, steroidogenic metabolon, and signaling proteins), trace the original archetype, and predict their biological functions by molecular phylogenetic and functional divergence analyses, protein homology modeling and molecular docking. Although most members of these complexes have a history of gene duplication and functional divergence during evolution, phylogenomic analysis revealed that all vertebrates have the same functional complex members, suggesting a common mechanism in the first step of steroidogenesis. An archetype of the complex was found in invertebrates. The data presented herein suggest that the cholesterol transport machinery is responsible for steroidogenesis among all vertebrates and is evolutionarily conserved throughout the entire animal kingdom. PMID:24124589

  7. 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

  8. Active polysomes in the axoplasm of the squid giant axon.

    PubMed

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

    1991-01-01

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

  9. Torsional Behavior of Axonal Microtubule Bundles

    PubMed Central

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

    2015-01-01

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

  10. 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

  11. 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

  12. Optically Resolving Individual Microtubules in Live Axons

    PubMed Central

    Mudrakola, Harsha V.; Zhang, Kai; Cui, Bianxiao

    2010-01-01

    Summary Microtubules are essential cytoskeletal tracks for cargo transportation in axons and also serve as the primary structural scaffold of neurons. Structural assembly, stability, and dynamics of axonal microtubules are of great interest for understanding neuronal functions and pathologies. However, microtubules are so densely packed in axons that their separations are well below the diffraction limit of light, which precludes using optical microscopy for live-cell studies. Here, we present a single-molecule imaging method capable of resolving individual microtubules in live axons. In our method, unlabeled microtubules are revealed by following individual axonal cargos that travel along them. We resolved more than six microtubules in a 1 μm diameter axon by real-time tracking of endosomes containing quantum dots. Our live-cell study also provided direct evidence that endosomes switch between microtubules while traveling along axons, which has been proposed to be the primary means for axonal cargos to effectively navigate through the crowded axoplasmic environment. PMID:19913478

  13. Importance of mitochondrial P(O2) in maximal O2 transport and utilization: a theoretical analysis.

    PubMed

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

    2013-12-01

    In previous calculations of how the O2 transport system limits .VO2(max), it was reasonably assumed that mitochondrial P(O2) (Pm(O2)) could be neglected (set to zero). However, in reality, Pm(O2) must exceed zero and the red cell to mitochondrion diffusion gradient may therefore be reduced, impairing diffusive transport of O2 and .VO2(max). Accordingly, we investigated the influence of Pm(O2) on these calculations by coupling previously used equations for O2 transport to one for mitochondrial respiration relating mitochondrial .VO2 to P(O2). This hyperbolic function, characterized by its P50 and V˙MAX, allowed Pm(O2) to become a model output (rather than set to zero as previously). Simulations using data from exercising normal subjects showed that at .VO2(max), Pm(O2) was usually <1mmHg, and that the effects on .VO2(max) were minimal. However, when O2 transport capacity exceeded mitochondrial V˙MAX, or if P50 were elevated,Pm(O2) often reached double digit values, thereby reducing the diffusion gradient and significantly decreasing .VO2(max).

  14. 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

  15. Iron transport by proteoliposomes containing mitochondrial F(1)F(0) ATP synthase isolated from rat heart.

    PubMed

    Kim, Misun; Song, Eunsook

    2010-04-01

    In this work, we present evidence of Fe(2+) transport by rat heart mitochondrial F(1)F(0) ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 degrees C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the alpha, beta, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg(+2), Ca(+2), Mn(+2), Zn(+2), Cu(+2), Fe(+3), and Al(+3) was observed. Moreover, Cu(+2), even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H(+). PMID:20100539

  16. 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.

  17. A crosstalk between Na⁺ channels, Na⁺/K⁺ pump and mitochondrial Na⁺ transporters controls glucose-dependent cytosolic and mitochondrial Na⁺ signals.

    PubMed

    Nita, Iulia I; Hershfinkel, Michal; Lewis, Eli C; Sekler, Israel

    2015-02-01

    Glucose-dependent cytosolic Na(+) influx in pancreatic islet β cells is mediated by TTX-sensitive Na(+) channels and is propagated into the mitochondria through the mitochondrial Na(+)/Ca(2+) exchanger, NCLX. Mitochondrial Na(+) transients are also controlled by the mitochondrial Na(+)/H(+) exchanger, NHE, while cytosolic Na(+) changes are governed by Na(+)/K(+) ATPase pump. The functional interaction between the Na(+) channels, Na(+)/K(+) ATPase pump and mitochondrial Na(+) transporters, NCLX and NHE, in mediating Na(+) signaling is poorly understood. Here, we combine fluorescent Na(+) imaging, pharmacological inhibition by TTX, ouabain and EIPA, with molecular control of NCLX expression, so as to investigate the crosstalk between Na(+) transporters on both the plasma membrane and the mitochondria. According to our results, glucose-dependent cytosolic Na(+) response was enhanced by ouabain and was followed by a rise in mitochondrial Na(+) signal. Silencing of NCLX expression using siNCLX, did not affect the glucose- or ouabain-dependent cytosolic rise in Na(+). In contrast, the ouabain-dependent rise in mitochondrial Na(+) was strongly suppressed by siNCLX. Furthermore, mitochondrial Na(+) influx rates were accelerated in cells treated with the Na(+)/H(+) exchanger inhibitor, EIPA or by combination of EIPA and ouabain. Similarly, TTX blocked the cytosolic and mitochondrial Na(+) responses, which were enhanced by ouabain or EIPA, respectively. Our results suggest that Na(+)/K(+) ATPase pump controls cytosolic glucose-dependent Na(+) rise, in a manner that is mediated by TTX-sensitive Na(+) channels and subsequent mitochondrial Na(+) uptake via NCLX. Furthermore, these results indicate that mitochondrial Na(+) influx via NCLX is antagonized by Na(+) efflux, which is mediated by the mitochondrial NHE; thus, the duration of mitochondrial Na(+) transients is set by the interplay between these pivotal transporters.

  18. Inhibition of mitochondrial pyruvate transport by zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina.

    PubMed

    Du, Jianhai; Cleghorn, Whitney M; Contreras, Laura; Lindsay, Ken; Rountree, Austin M; Chertov, Andrei O; Turner, Sally J; Sahaboglu, Ayse; Linton, Jonathan; Sadilek, Martin; Satrústegui, Jorgina; Sweet, Ian R; Paquet-Durand, François; Hurley, James B

    2013-12-13

    Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing (13)C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.

  19. Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina*

    PubMed Central

    Du, Jianhai; Cleghorn, Whitney M.; Contreras, Laura; Lindsay, Ken; Rountree, Austin M.; Chertov, Andrei O.; Turner, Sally J.; Sahaboglu, Ayse; Linton, Jonathan; Sadilek, Martin; Satrústegui, Jorgina; Sweet, Ian R.; Paquet-Durand, François; Hurley, James B.

    2013-01-01

    Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing 13C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia. PMID:24187136

  20. 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

  1. Axonal structure and function after axolemmal leakage in the squid giant axon.

    PubMed

    Gallant, P E; Galbraith, J A

    1997-11-01

    Membrane leakage is a common consequence of traumatic nerve injury. In order to measure the early secondary effects of different levels of membrane leakage on axonal structure and function we studied the squid giant axon after electroporation at field strengths of 0.5, 1.0, 1.6, or 3.3 kV/cm. Immediately after mild electroporation at 0.5 kV/cm, 40% of the axons had no action potentials, but by 1 h all of the mildly electroporated axons had recovered their action potentials. Many large organelles (mitochondria) were swollen, however, and their transport was reduced by 62% 1 h after this mild electroporation. One hour after moderate electroporation at 1.0 kV/cm, most of the axons had no action potentials, most large organelles were swollen, and their transport was reduced by 98%, whereas small organelle transport was reduced by 75%. Finally at severe electroporation levels of 1.65-3.0 kV/cm all conduction and transport was lost and the gel-like axoplasmic structure was clumped or liquefied. The structural damage and transport block seen after severe and moderate poration were early secondary injuries that could be prevented by placing the porated axons in an intracellular-type medium (low in Ca2+, Na+, and Cl-) immediately after poration. In moderately, but not severely, porated axons this protection of organelle transport and structure persisted, and action potential conduction returned when the axons were returned to the previously injurious extracellular-type medium. This suggests that the primary damage, the axolemmal leak, was repaired while the moderately porated axons were in the protective intracellular-type medium.

  2. Dynamic microtubule organization and mitochondrial transport are regulated by distinct Kinesin-1 pathways

    PubMed Central

    Melkov, Anna; Simchoni, Yasmin; Alcalay, Yehonatan; Abdu, Uri

    2015-01-01

    ABSTRACT The microtubule (MT) plus-end motor kinesin heavy chain (Khc) is well known for its role in long distance cargo transport. Recent evidence showed that Khc is also required for the organization of the cellular MT network by mediating MT sliding. We found that mutations in Khc and the gene of its adaptor protein, kinesin light chain (Klc) resulted in identical bristle morphology defects, with the upper part of the bristle being thinner and flatter than normal and failing to taper towards the bristle tip. We demonstrate that bristle mitochondria transport requires Khc but not Klc as a competing force to dynein heavy chain (Dhc). Surprisingly, we demonstrate for the first time that Dhc is the primary motor for both anterograde and retrograde fast mitochondria transport. We found that the upper part of Khc and Klc mutant bristles lacked stable MTs. When following dynamic MT polymerization via the use of GFP-tagged end-binding protein 1 (EB1), it was noted that at Khc and Klc mutant bristle tips, dynamic MTs significantly deviated from the bristle parallel growth axis, relative to wild-type bristles. We also observed that GFP-EB1 failed to concentrate as a focus at the tip of Khc and Klc mutant bristles. We propose that the failure of bristle tapering is due to defects in directing dynamic MTs at the growing tip. Thus, we reveal a new function for Khc and Klc in directing dynamic MTs during polarized cell growth. Moreover, we also demonstrate a novel mode of coordination in mitochondrial transport between Khc and Dhc. PMID:26581590

  3. The Inter-Relationship of Ascorbate Transport, Metabolism and Mitochondrial, Plastidic Respiration

    PubMed Central

    Bánhegyi, Gábor; Asard, Han

    2013-01-01

    Abstract Significance: Ascorbate, this multifaceted small molecular weight carbohydrate derivative, plays important roles in a range of cellular processes in plant cells, from the regulation of cell cycle, through cell expansion and senescence. Beyond these physiological functions, ascorbate has a critical role in responses to abiotic stresses, such as high light, high salinity, or drought. The biosynthesis, recycling, and intracellular transport are important elements of the balancing of ascorbate level to the always-changing conditions and demands. Recent Advances: A bidirectional tight relationship was described between ascorbate biosynthesis and the mitochondrial electron transfer chain (mETC), since L-galactono-1,4-lactone dehydrogenase (GLDH), the enzyme catalyzing the ultimate step of ascorbate biosynthesis, uses oxidized cytochrome c as the only electron acceptor and has a role in the assembly of Complex I. A similar bidirectional relationship was revealed between the photosynthetic apparatus and ascorbate biosynthesis since the electron flux through the photosynthetic ETC affects the biosynthesis of ascorbate and the level of ascorbate could affect photosynthesis. Critical Issues: The details of this regulatory network of photosynthetic electron transfer, respiratory electron transfer, and ascorbate biosynthesis are still not clear, as are the potential regulatory role and the regulation of intracellular ascorbate transport and fluxes. Future Directions: The elucidation of the role of ascorbate as an important element of the network of photosynthetic, respiratory ETC and tricarboxylic acid cycle will contribute to understanding plant cell responses to different stress conditions. Antioxid. Redox Signal. 19, 1036–1044. PMID:23259603

  4. Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells.

    PubMed

    Ruas, Juliana S; Siqueira-Santos, Edilene S; Amigo, Ignacio; Rodrigues-Silva, Erika; Kowaltowski, Alicia J; Castilho, Roger F

    2016-01-01

    The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore

  5. Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells.

    PubMed

    Ruas, Juliana S; Siqueira-Santos, Edilene S; Amigo, Ignacio; Rodrigues-Silva, Erika; Kowaltowski, Alicia J; Castilho, Roger F

    2016-01-01

    The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore

  6. Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells

    PubMed Central

    Ruas, Juliana S.; Siqueira-Santos, Edilene S.; Amigo, Ignacio; Rodrigues-Silva, Erika; Kowaltowski, Alicia J.; Castilho, Roger F.

    2016-01-01

    The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore

  7. Axonal regeneration in zebrafish.

    PubMed

    Becker, Thomas; Becker, Catherina G

    2014-08-01

    In contrast to mammals, fish and amphibia functionally regenerate axons in the central nervous system (CNS). The strengths of the zebrafish model, that is, transgenics and mutant availability, ease of gene expression analysis and manipulation and optical transparency of larvae lend themselves to the analysis of successful axonal regeneration. Analyses in larval and adult zebrafish suggest a high intrinsic capacity for axon regrowth, yet signaling pathways employed in axonal growth and pathfinding are similar to those in mammals. However, the lesioned CNS environment in zebrafish shows remarkably little scarring or expression of inhibitory molecules and regenerating axons use molecular cues in the environment to successfully navigate to their targets. Future zebrafish research, including screening techniques, will complete our picture of the mechanisms behind successful CNS axon regeneration in this vertebrate model organism.

  8. Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.

    PubMed

    Ruprecht, Jonathan J; Hellawell, Alex M; Harding, Marilyn; Crichton, Paul G; McCoy, Airlie J; Kunji, Edmund R S

    2014-01-28

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix. The carrier cycles by an unresolved mechanism between the cytoplasmic state, in which the carrier accepts ADP from the cytoplasm, and the matrix state, in which it accepts ATP from the mitochondrial matrix. Here we present the structures of the yeast ADP/ATP carriers Aac2p and Aac3p in the cytoplasmic state. The carriers have three domains and are closed at the matrix side by three interdomain salt-bridge interactions, one of which is braced by a glutamine residue. Glutamine braces are conserved in mitochondrial carriers and contribute to an energy barrier, preventing the conversion to the matrix state unless substrate binding occurs. At the cytoplasmic side a second salt-bridge network forms during the transport cycle, as demonstrated by functional analysis of mutants with charge-reversed networks. Analyses of the domain structures and properties of the interdomain interfaces indicate that interconversion between states involves movement of the even-numbered α-helices across the surfaces of the odd-numbered α-helices by rotation of the domains. The odd-numbered α-helices have an L-shape, with proline or serine residues at the kinks, which functions as a lever-arm, coupling the substrate-induced disruption of the matrix network to the formation of the cytoplasmic network. The simultaneous movement of three domains around a central translocation pathway constitutes a unique mechanism among transport proteins. These findings provide a structural description of transport by mitochondrial carrier proteins, consistent with an alternating-access mechanism.

  9. Molecular Determinants of the Axonal mRNA Transcriptome

    PubMed Central

    Gomes, Cynthia; Merianda, Tanuja T.; Lee, Seung Joon; Yoo, Soonmoon; Twiss, Jeffery L.

    2014-01-01

    Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in cell body responses to axotomy. Recent studies have begun to identify the protein products that contribute to these autonomous responses of axons. In the peripheral nervous system, intra-axonal protein synthesis has been implicated in the localized in vivo responses to neuropathic stimuli, and there is emerging evidence for protein synthesis in CNS axons in vivo. Despite that hundreds of mRNAs have now been shown to localize into the axonal compartment, knowledge of what RNA binding proteins are responsible for this is quite limited. Here, we review the current state of knowledge of RNA transport mechanisms, and highlight recently uncovered mechanisms for dynamically altering the axonal transcriptome. Both changes in the levels or activities of components of the RNA transport apparatus and alterations in transcription of transported mRNAs can effectively shift the axonal mRNA population. Consistent with this, the axonal RNA population shifts with development, with changes in growth state, and in response to extracellular stimulation. Each of these events must impact the transcriptional and transport apparatuses of the neuron, thus directly and indirectly modifying the axonal transcriptome. PMID:23959706

  10. Mitochondrial Cardiomyopathies

    PubMed Central

    El-Hattab, Ayman W.; Scaglia, Fernando

    2016-01-01

    Mitochondria are found in all nucleated human cells and perform various essential functions, including the generation of cellular energy. Mitochondria are under dual genome control. Only a small fraction of their proteins are encoded by mitochondrial DNA (mtDNA), whereas more than 99% of them are encoded by nuclear DNA (nDNA). Mutations in mtDNA or mitochondria-related nDNA genes result in mitochondrial dysfunction leading to insufficient energy production required to meet the needs for various organs, particularly those with high energy requirements, including the central nervous system, skeletal and cardiac muscles, kidneys, liver, and endocrine system. Because cardiac muscles are one of the high energy demanding tissues, cardiac involvement occurs in mitochondrial diseases with cardiomyopathies being one of the most frequent cardiac manifestations found in these disorders. Cardiomyopathy is estimated to occur in 20–40% of children with mitochondrial diseases. Mitochondrial cardiomyopathies can vary in severity from asymptomatic status to severe manifestations including heart failure, arrhythmias, and sudden cardiac death. Hypertrophic cardiomyopathy is the most common type; however, mitochondrial cardiomyopathies might also present as dilated, restrictive, left ventricular non-compaction, and histiocytoid cardiomyopathies. Cardiomyopathies are frequent manifestations of mitochondrial diseases associated with defects in electron transport chain complexes subunits and their assembly factors, mitochondrial transfer RNAs, ribosomal RNAs, ribosomal proteins, translation factors, mtDNA maintenance, and coenzyme Q10 synthesis. Other mitochondrial diseases with cardiomyopathies include Barth syndrome, Sengers syndrome, TMEM70-related mitochondrial complex V deficiency, and Friedreich ataxia. PMID:27504452

  11. Mitochondrial Cardiomyopathies.

    PubMed

    El-Hattab, Ayman W; Scaglia, Fernando

    2016-01-01

    Mitochondria are found in all nucleated human cells and perform various essential functions, including the generation of cellular energy. Mitochondria are under dual genome control. Only a small fraction of their proteins are encoded by mitochondrial DNA (mtDNA), whereas more than 99% of them are encoded by nuclear DNA (nDNA). Mutations in mtDNA or mitochondria-related nDNA genes result in mitochondrial dysfunction leading to insufficient energy production required to meet the needs for various organs, particularly those with high energy requirements, including the central nervous system, skeletal and cardiac muscles, kidneys, liver, and endocrine system. Because cardiac muscles are one of the high energy demanding tissues, cardiac involvement occurs in mitochondrial diseases with cardiomyopathies being one of the most frequent cardiac manifestations found in these disorders. Cardiomyopathy is estimated to occur in 20-40% of children with mitochondrial diseases. Mitochondrial cardiomyopathies can vary in severity from asymptomatic status to severe manifestations including heart failure, arrhythmias, and sudden cardiac death. Hypertrophic cardiomyopathy is the most common type; however, mitochondrial cardiomyopathies might also present as dilated, restrictive, left ventricular non-compaction, and histiocytoid cardiomyopathies. Cardiomyopathies are frequent manifestations of mitochondrial diseases associated with defects in electron transport chain complexes subunits and their assembly factors, mitochondrial transfer RNAs, ribosomal RNAs, ribosomal proteins, translation factors, mtDNA maintenance, and coenzyme Q10 synthesis. Other mitochondrial diseases with cardiomyopathies include Barth syndrome, Sengers syndrome, TMEM70-related mitochondrial complex V deficiency, and Friedreich ataxia. PMID:27504452

  12. The central role of mitochondria in axonal degeneration in multiple sclerosis.

    PubMed

    Campbell, Graham R; Worrall, Joseph T; Mahad, Don J

    2014-12-01

    Neurodegeneration in multiple sclerosis (MS) is related to inflammation and demyelination. In acute MS lesions and experimental autoimmune encephalomyelitis focal immune attacks damage axons by injuring axonal mitochondria. In progressive MS, however, axonal damage occurs in chronically demyelinated regions, myelinated regions and also at the active edge of slowly expanding chronic lesions. How axonal energy failure occurs in progressive MS is incompletely understood. Recent studies show that oligodendrocytes supply lactate to myelinated axons as a metabolic substrate for mitochondria to generate ATP, a process which will be altered upon demyelination. In addition, a number of studies have identified mitochondrial abnormalities within neuronal cell bodies in progressive MS, leading to a deficiency of mitochondrial respiratory chain complexes or enzymes. Here, we summarise the mitochondrial abnormalities evident within neurons and discuss how these grey matter mitochondrial abnormalities may increase the vulnerability of axons to degeneration in progressive MS. Although neuronal mitochondrial abnormalities will culminate in axonal degeneration, understanding the different contributions of mitochondria to the degeneration of myelinated and demyelinated axons is an important step towards identifying potential therapeutic targets for progressive MS.

  13. Delayed Feedback Model of Axonal Length Sensing

    PubMed Central

    Karamched, Bhargav R.; Bressloff, Paul C.

    2015-01-01

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

  14. An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis.

    PubMed

    Birsoy, Kıvanç; Wang, Tim; Chen, Walter W; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M

    2015-07-30

    The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. PMID:26232224

  15. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis

    PubMed Central

    Birsoy, Kıvanç; Wang, Tim; Chen, Walter; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M.

    2015-01-01

    Summary The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. PMID:26232224

  16. Impaired mitochondrial energy production and ABC transporter function-A crucial interconnection in dementing proteopathies of the brain.

    PubMed

    Pahnke, Jens; Fröhlich, Christina; Krohn, Markus; Schumacher, Toni; Paarmann, Kristin

    2013-10-01

    Ageing is the main risk factor for the development of dementing neurodegenerative diseases (NDs) and it is accompanied by the accumulation of variations in mitochondrial DNA. The resulting tissue-specific alterations in ATP production and availability cause deteriorations of cerebral clearance mechanisms that are important for the removal of toxic peptides and its aggregates. ABC transporters were shown to be the most important exporter superfamily for toxic peptides, e.g. β-amyloid and α-synuclein. Their activity is highly dependent on the availability of ATP and forms a directed energy-exporter network, linking decreased mitochondrial function with highly impaired ABC transporter activity and disease progression. In this paper, we describe a network based on interactions between ageing, energy metabolism, regeneration, accumulation of toxic peptides and the development of proteopathies of the brain with a focus on Alzheimer's disease (AD). Additionally, we provide new experimental evidence for interactions within this network in regenerative processes in AD.

  17. Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights.

    PubMed

    Gawryluk, Ryan M R; Chisholm, Kenneth A; Pinto, Devanand M; Gray, Michael W

    2012-11-01

    The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits.

  18. Kalkitoxin Inhibits Angiogenesis, Disrupts Cellular Hypoxic Signaling, and Blocks Mitochondrial Electron Transport in Tumor Cells

    PubMed Central

    Morgan, J. Brian; Liu, Yang; Coothankandaswamy, Veena; Mahdi, Fakhri; Jekabsons, Mika B.; Gerwick, William H.; Valeriote, Frederick A.; Zhou, Yu-Dong; Nagle, Dale G.

    2015-01-01

    The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-d-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells. PMID:25803180

  19. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

    PubMed

    Keller, J N; Mark, R J; Bruce, A J; Blanc, E; Rothstein, J D; Uchida, K; Waeg, G; Mattson, M P

    1997-10-01

    Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4

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

    PubMed Central

    2012-01-01

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

  1. Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism

    PubMed Central

    Giudetti, Anna M.; Stanca, Eleonora; Siculella, Luisa; Gnoni, Gabriele V.; Damiano, Fabrizio

    2016-01-01

    The transport of solutes across the inner mitochondrial membrane is catalyzed by a family of nuclear-encoded membrane-embedded proteins called mitochondrial carriers (MCs). The citrate carrier (CiC) and the carnitine/acylcarnitine transporter (CACT) are two members of the MCs family involved in fatty acid metabolism. By conveying acetyl-coenzyme A, in the form of citrate, from the mitochondria to the cytosol, CiC contributes to fatty acid and cholesterol synthesis; CACT allows fatty acid oxidation, transporting cytosolic fatty acids, in the form of acylcarnitines, into the mitochondrial matrix. Fatty acid synthesis and oxidation are inversely regulated so that when fatty acid synthesis is activated, the catabolism of fatty acids is turned-off. Malonyl-CoA, produced by acetyl-coenzyme A carboxylase, a key enzyme of cytosolic fatty acid synthesis, represents a regulator of both metabolic pathways. CiC and CACT activity and expression are regulated by different nutritional and hormonal conditions. Defects in the corresponding genes have been directly linked to various human diseases. This review will assess the current understanding of CiC and CACT regulation; underlining their roles in physio-pathological conditions. Emphasis will be placed on the molecular basis of the regulation of CiC and CACT associated with fatty acid metabolism. PMID:27231907

  2. Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons.

    PubMed Central

    Mohr, E; Morris, J F; Richter, D

    1995-01-01

    It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7753814

  3. cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells.

    PubMed

    Castillo, Ana Fernanda; Cornejo Maciel, Fabiana; Castilla, Rocío; Duarte, Alejandra; Maloberti, Paula; Paz, Cristina; Podestá, Ernesto J

    2006-11-01

    We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.

  4. Axonal GABAA receptors.

    PubMed

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

    2008-09-01

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

  5. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial glutathione.

    PubMed

    García-Ruiz, C; Colell, A; Marí, M; Morales, A; Fernández-Checa, J C

    1997-04-25

    Ceramide is a sphingolipid that is generated in the signaling of inflammatory cytokines such as tumor necrosis factor (TNF), which exerts many functional roles depending on the cell type where it is produced. Since TNF cytotoxicity is mediated by overproduction of reactive oxygen species from mitochondria, we have examined the role of ceramide in generation of oxidative stress in isolated rat liver mitochondria. The present studies demonstrate that addition of N-acetylsphingosine (C2-ceramide) to mitochondria led to an increase of fluorescence of dihydrorhodamine 123 or dichlorofluorescein-stained mitochondria, indicating formation of hydrogen peroxide. Such effect was significant at 0.25 microM and maximal at 1-5 microM C2, decreasing at greater concentrations. This inductive effect of ceramide was mimicked by N-hexanoylsphingosine at the same concentration range, whereas the immediate precursor of C2, C2-dihydroceramide increased hydrogen peroxide at 1-5 microM. Sphingosine generated hydrogen peroxide at concentrations >/=10 microM, whereas diacylglycerol failed to increase hydrogen peroxide. The increase in hydrogen peroxide induced by C2 was not triggered by mitochondrial permeability transition as C2 did not induce mitochondrial swelling. Blocking electron transport chain at complex I and II prevented the increase in hydrogen peroxide induced by C2; however, interruption of electron flow at complex III by antimycin A potentiated the inductive effect of C2. Depletion of matrix GSH prior to exposure to ceramide resulted in a potentiated increase (2-fold) of hydrogen peroxide generation, leading to lipid peroxidation and loss of activity of respiratory chain complex IV compared with GSH-repleted mitochondria. Mitochondria isolated from TNF-treated cells showed an increase (2-3-fold) in the amount of ceramide compared with mitochondria from untreated cells. These results suggest that mitochondria are a target of ceramide produced in the signaling of TNF whose

  6. Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy

    PubMed Central

    Ahmad, Tanveer; Mukherjee, Shravani; Pattnaik, Bijay; Kumar, Manish; Singh, Suchita; Kumar, Manish; Rehman, Rakhshinda; Tiwari, Brijendra K; Jha, Kumar A; Barhanpurkar, Amruta P; Wani, Mohan R; Roy, Soumya S; Mabalirajan, Ulaganathan; Ghosh, Balaram; Agrawal, Anurag

    2014-01-01

    There is emerging evidence that stem cells can rejuvenate damaged cells by mitochondrial transfer. Earlier studies show that epithelial mitochondrial dysfunction is critical in asthma pathogenesis. Here we show for the first time that Miro1, a mitochondrial Rho-GTPase, regulates intercellular mitochondrial movement from mesenchymal stem cells (MSC) to epithelial cells (EC). We demonstrate that overexpression of Miro1 in MSC (MSCmiroHi) leads to enhanced mitochondrial transfer and rescue of epithelial injury, while Miro1 knockdown (MSCmiroLo) leads to loss of efficacy. Treatment with MSCmiroHi was associated with greater therapeutic efficacy, when compared to control MSC, in mouse models of rotenone (Rot) induced airway injury and allergic airway inflammation (AAI). Notably, airway hyperresponsiveness and remodeling were reversed by MSCmiroHi in three separate allergen-induced asthma models. In a human in vitro system, MSCmiroHi reversed mitochondrial dysfunction in bronchial epithelial cells treated with pro-inflammatory supernatant of IL-13-induced macrophages. Anti-inflammatory MSC products like NO, TGF-β, IL-10 and PGE2, were unchanged by Miro1 overexpression, excluding non-specific paracrine effects. In summary, Miro1 overexpression leads to increased stem cell repair. PMID:24431222

  7. Genetic labeling of both the axons of transduced, glutamatergic neurons in rat postrhinal cortex and their postsynaptic neurons in other neocortical areas by herpes simplex virus vectors that coexpress an axon-targeted β-galactosidase and wheat germ agglutinin from a vesicular glutamate transporter-1 promoter.

    PubMed

    Zhang, Guo-rong; Cao, Haiyan; Li, Xu; Zhao, Hua; Geller, Alfred I

    2010-11-18

    Neuronal circuits comprise the foundation for neuronal physiology and synaptic plasticity, and thus for consequent behaviors and learning, but our knowledge of neocortical circuits is incomplete. Mapping neocortical circuits is a challenging problem because these circuits contain large numbers of neurons, a high density of synapses, and numerous classes and subclasses of neurons that form many different types of synapses. Expression of specific genetic tracers in small numbers of specific subclasses of neocortical neurons has the potential to map neocortical circuits. Suitable genetic tracers have been established in neurons in subcortical areas, but application to neocortical circuits has been limited. Enabling this approach, Herpes Simplex Virus (HSV-1) plasmid (amplicon) vectors can transduce small numbers of neurons in a specific neocortical area. Further, expression of a particular genetic tracer can be restricted to specific subclasses of neurons; in particular, the vesicular glutamate transporter-1 (VGLUT1) promoter supports expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex. Here, we show that expression of an axon-targeted β-galactosidase (β-gal) from such vectors supports mapping specific commissural and associative projections of the transduced neurons in POR cortex. Further, coexpression of wheat germ agglutinin (WGA) and an axon-targeted β-gal supports mapping both specific projections of the transduced neurons and identifying specific postsynaptic neurons for the transduced neurons. The neocortical circuit mapping capabilities developed here may support mapping specific neocortical circuits that have critical roles in cognitive learning.

  8. Axons take a dive

    PubMed Central

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

    2015-01-01

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

  9. Axonal bleb recording.

    PubMed

    Hu, Wenqin; Shu, Yousheng

    2012-08-01

    Patch-clamp recording requires direct accessibility of the cell membrane to patch pipettes and allows the investigation of ion channel properties and functions in specific cellular compartments. The cell body and relatively thick dendrites are the most accessible compartments of a neuron, due to their large diameters and therefore great membrane surface areas. However, axons are normally inaccessible to patch pipettes because of their thin structure; thus studies of axon physiology have long been hampered by the lack of axon recording methods. Recently, a new method of patch-clamp recording has been developed, enabling direct and tight-seal recording from cortical axons. These recordings are performed at the enlarged structure (axonal bleb) formed at the cut end of an axon after slicing procedures. This method has facilitated studies of the mechanisms underlying the generation and propagation of the main output signal, the action potential, and led to the finding that cortical neurons communicate not only in action potential-mediated digital mode but also in membrane potential-dependent analog mode. PMID:22833034

  10. Microchannels as axonal amplifiers.

    PubMed

    Fitzgerald, James J; Lacour, Stéphanie P; McMahon, Stephen B; Fawcett, James W

    2008-03-01

    An implantable neural interface capable of reliable long-term high-resolution recording from peripheral nerves has yet to be developed. Device design is challenging because extracellular axonal signals are very small, decay rapidly with distance from the axon, and in myelinated fibres are concentrated close to nodes of Ranvier, which are around 1 mum long and spaced several hundred micrometers apart. We present a finite element model examining the electrical behavior of axons in microchannels, and demonstrate that confining axons in such channels substantially amplifies the extracellular signal. For example, housing a 10-microm myelinated axon in a 1-cm-long channel with a 1000-microm(2) cross section is predicted to generate a peak extracellular voltage of over 10 mV. Furthermore, there is little radial signal decay within the channel, and a smooth axial variation of signal amplitude along the channel, irrespective of node location. Additional benefits include a greater extracellular voltage generated by large myelinated fibres compared to small unmyelinated axons, and the reduction of gain to unity at the end of the channel which ensures that there can be no crosstalk with electrodes in other channels nearby. A microchannel architecture seems well suited to the requirements of a peripheral nerve interface.

  11. Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in Saccharomyces cerevisiae.

    PubMed

    Tian, Siqi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2015-01-01

    To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.

  12. Alterations in the mitochondrial alternative NAD(P)H Dehydrogenase NDB4 lead to changes in mitochondrial electron transport chain composition, plant growth and response to oxidative stress.

    PubMed

    Smith, Chevaun; Barthet, Michelle; Melino, Vanessa; Smith, Penelope; Day, David; Soole, Kathleen

    2011-07-01

    The branched respiratory electron transport chain of plants contains a non-phosphorylating alternative pathway consisting of type II NAD(P)H dehydrogenases on both sides of the inner membrane linked through the ubiquinone pool to an alternative oxidase (AOX). T-DNA and RNA interference (RNAi) were used to reduce gene expression to characterize the external NAD(P)H dehydrogenase NDB4 in Arabidopsis. The ndb4 lines showed different levels of suppression of NDB4 protein, leading to increases in NBD2 and AOX1a mRNA and protein levels in all lines. These changes were associated with lower reactive oxygen species formation and an altered phenotype, including changes in growth rate, root : shoot ratios and leaf area. The general growth pattern for the ndb4 mutants was decreased leaf area early in development (6-15 d) followed by a prompt subsequent increase in leaf area that exceeded the leaf area of the wild type by maturity (the 10-12 rosette stage). This pattern was most evident for the RNAi lines that had increased mitochondrial electron transport capacity. The RNAi lines also exhibited better tolerance to salinity stress, with better growth rates and lower shoot Na⁺ content compared with controls when grown under saline conditions. We hypothesize that these differences reflect the enhanced expression of NDB2 and AOX in the ndb4 mutant plants.

  13. Mitochondrial aquaporin-8-mediated hydrogen peroxide transport is essential for teleost spermatozoon motility

    PubMed Central

    Chauvigné, François; Boj, Mónica; Finn, Roderick Nigel; Cerdà, Joan

    2015-01-01

    Reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), cause oxidative cell damage and inhibit sperm function. In most oviparous fishes that spawn in seawater (SW), spermatozoa may be exposed to harmful ROS loads associated with the hyperosmotic stress of axonemal activation and ATP synthesis from mitochondrial oxidative phosphorylation. However, it is not known how marine spermatozoa can cope with the increased ROS levels to maintain flagellar motility. Here, we show that a marine teleost orthologue of human aquaporin-8, termed Aqp8b, is rapidly phosphorylated and inserted into the inner mitochondrial membrane of SW-activated spermatozoa, where it facilitates H2O2 efflux from this compartment. When Aqp8b intracellular trafficking and mitochondrial channel activity are immunologically blocked in activated spermatozoa, ROS levels accumulate in the mitochondria leading to mitochondrial membrane depolarisation, the reduction of ATP production, and the progressive arrest of sperm motility. However, the decreased sperm vitality underlying Aqp8b loss of function is fully reversed in the presence of a mitochondria-targeted antioxidant. These findings reveal a previously unknown detoxification mechanism in spermatozoa under hypertonic conditions, whereby mitochondrial Aqp8b-mediated H2O2 efflux permits fuel production and the maintenance of flagellar motility. PMID:25586329

  14. Sodium Channels, Mitochondria, and Axonal Degeneration in Peripheral Neuropathy.

    PubMed

    Persson, Anna-Karin; Hoeijmakers, Janneke G J; Estacion, Mark; Black, Joel A; Waxman, Stephen G

    2016-05-01

    Peripheral neuropathy results from damage to peripheral nerves and is often accompanied by pain in affected limbs. Treatment represents an unmet medical need and a thorough understanding of the mechanisms underlying axonal injury is needed. Longer nerve fibers tend to degenerate first (length-dependence), and patients carrying pathogenic mutations throughout life usually become symptomatic in mid- or late-life (time-dependence). The activity of voltage-gated sodium channels can contribute to axonal injury and sodium channel gain-of-function mutations have been linked to peripheral neuropathy. Recent studies have implicated sodium channel activity, mitochondrial compromise, and reverse-mode Na(+)/Ca(2+) exchange in time- and length-dependent axonal injury. Elucidation of molecular mechanisms underlying axonal injury in peripheral neuropathy may provide new therapeutic strategies for this painful and debilitating condition.

  15. Mitochondrial cytopathies.

    PubMed

    El-Hattab, Ayman W; Scaglia, Fernando

    2016-09-01

    Mitochondria are found in all nucleated human cells and perform a variety of essential functions, including the generation of cellular energy. Most of mitochondrial proteins are encoded by the nuclear DNA (nDNA) whereas a very small fraction is encoded by the mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondria-related nDNA genes can result in mitochondrial dysfunction which leads to a wide range of cellular perturbations including aberrant calcium homeostasis, excessive reactive oxygen species production, dysregulated apoptosis, and insufficient energy generation to meet the needs of various organs, particularly those with high energy demand. Impaired mitochondrial function in various tissues and organs results in the multi-organ manifestations of mitochondrial diseases including epilepsy, intellectual disability, skeletal and cardiac myopathies, hepatopathies, endocrinopathies, and nephropathies. Defects in nDNA genes can be inherited in an autosomal or X-linked manners, whereas, mtDNA is maternally inherited. Mitochondrial diseases can result from mutations of nDNA genes encoding subunits of the electron transport chain complexes or their assembly factors, proteins associated with the mitochondrial import or networking, mitochondrial translation factors, or proteins involved in mtDNA maintenance. MtDNA defects can be either point mutations or rearrangements. The diagnosis of mitochondrial disorders can be challenging in many cases and is based on clinical recognition, biochemical screening, histopathological studies, functional studies, and molecular genetic testing. Currently, there are no satisfactory therapies available for mitochondrial disorders that significantly alter the course of the disease. Therapeutic options include symptomatic treatment, cofactor supplementation, and exercise. PMID:26996063

  16. Concepts for regulation of axon integrity by enwrapping glia

    PubMed Central

    Beirowski, Bogdan

    2013-01-01

    Long axons and their enwrapping glia (EG; Schwann cells (SCs) and oligodendrocytes (OLGs)) form a unique compound structure that serves as conduit for transport of electric and chemical information in the nervous system. The peculiar cytoarchitecture over an enormous length as well as its substantial energetic requirements make this conduit particularly susceptible to detrimental alterations. Degeneration of long axons independent of neuronal cell bodies is observed comparatively early in a range of neurodegenerative conditions as a consequence of abnormalities in SCs and OLGs . This leads to the most relevant disease symptoms and highlights the critical role that these glia have for axon integrity, but the underlying mechanisms remain elusive. The quest to understand why and how axons degenerate is now a crucial frontier in disease-oriented research. This challenge is most likely to lead to significant progress if the inextricable link between axons and their flanking glia in pathological situations is recognized. In this review I compile recent advances in our understanding of the molecular programs governing axon degeneration, and mechanisms of EG’s non-cell autonomous impact on axon-integrity. A particular focus is placed on emerging evidence suggesting that EG nurture long axons by virtue of their intimate association, release of trophic substances, and neurometabolic coupling. The correction of defects in these functions has the potential to stabilize axons in a variety of neuronal diseases in the peripheral nervous system and central nervous system (PNS and CNS). PMID:24391540

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  20. Mitochondrial phosphate transport. N-ethylmaleimide insensitivity correlates with absence of beef heart-like Cys42 from the Saccharomyces cerevisiae phosphate transport protein.

    PubMed

    Guérin, B; Bukusoglu, C; Rakotomanana, F; Wohlrab, H

    1990-11-15

    The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.

  1. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

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

  2. Use of DCPIP in a Colorimetric Method to Investigate Electron Transport in Crude Heart Mitochondrial Extracts.

    ERIC Educational Resources Information Center

    Myers, Alan

    1990-01-01

    A method of obtaining a crude mitochondrial extract from sheep and beef heart is described. The respiratory substrates succinate and malate are examined as well as the inhibitory effect of malonate. An interface with a microcomputer for plotting colorimeter results is discussed. (Author/CW)

  3. Electrophysiology of Axonal Constrictions

    NASA Astrophysics Data System (ADS)

    Johnson, Christopher; Jung, Peter; Brown, Anthony

    2013-03-01

    Axons of myelinated neurons are constricted at the nodes of Ranvier, where they are directly exposed to the extracellular space and where the vast majority of the ion channels are located. These constrictions are generated by local regulation of the kinetics of neurofilaments the most important cytoskeletal elements of the axon. In this paper we discuss how this shape affects the electrophysiological function of the neuron. Specifically, although the nodes are short (about 1 μm) in comparison to the distance between nodes (hundreds of μm) they have a substantial influence on the conduction velocity of neurons. We show through computational modeling that nodal constrictions (all other features such as numbers of ion channels left constant) reduce the required fiber diameter for a given target conduction velocity by up to 50% in comparison to an unconstricted axon. We further show that the predicted optimal fiber morphologies closely match reported fiber morphologies. Supported by The National Science Foundation (IOS 1146789)

  4. A novel method for producing mono-biotinylated, biologically active neurotrophic factors: an essential reagent for single molecule study of axonal transport.

    PubMed

    Sung, Kijung; Maloney, Michael T; Yang, Jingkun; Wu, Chengbiao

    2011-09-15

    In this report, we describe a novel method for producing mature and biologically active mono-biotinylated nerve growth factors (mBtNGF) that can be used for single molecule studies of real-time movement of neurotrophins within axons of neurons. We inserted an AviTag sequence into the C-terminal of the full length mouse preproNGF cDNA and cloned the fusion construct into a pcDNA3.1 mammalian expression vector. We also subcloned the Escherichia coli biotin ligase, BirA, into a pcDNA3.1 vector. These two plasmids were then transiently co-expressed in HEK293FT cells. As a result, the AviTag located in the C-terminal of preproNGF was selectively ligated to a single biotin by BirA. The prepro sequence of NGF was subsequently cleaved within the cell. Mature mono-biotinylated NGF (mBtNGF) was secreted into cell culture media and was purified using Ni resin. We carried out activity assays and our results showed that mBtNGF retained biological activities that were comparable to normal NGF purified from mouse sub maxillary glands. We further verified the biotinylation efficiency of mBtNGF and the level of non-biotinylated NGF was virtually undetectable in the final preparation. Finally, by conjugating to quantum-dot streptavidin, mBtNGF was successfully used for single molecule study of axonal NGF trafficking in neurons.

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

    PubMed Central

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

    2015-01-01

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

  6. The control of ionized calcium in squid axons

    PubMed Central

    Requena, J.; DiPolo, R; Brinley, FJ; Mullins, LJ

    1977-01-01

    Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 μmol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 μmol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 μmol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater. PMID:894259

  7. Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism.

    PubMed

    Saab, Aiman S; Tzvetavona, Iva D; Trevisiol, Andrea; Baltan, Selva; Dibaj, Payam; Kusch, Kathrin; Möbius, Wiebke; Goetze, Bianka; Jahn, Hannah M; Huang, Wenhui; Steffens, Heinz; Schomburg, Eike D; Pérez-Samartín, Alberto; Pérez-Cerdá, Fernando; Bakhtiari, Davood; Matute, Carlos; Löwel, Siegrid; Griesinger, Christian; Hirrlinger, Johannes; Kirchhoff, Frank; Nave, Klaus-Armin

    2016-07-01

    Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons. PMID:27292539

  8. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization

    PubMed Central

    Gumy, Laura F.; Yeo, Giles S.H.; Tung, Yi-Chun Loraine; Zivraj, Krishna H.; Willis, Dianna; Coppola, Giovanni; Lam, Brian Y.H.; Twiss, Jeffery L.; Holt, Christine E.; Fawcett, James W.

    2011-01-01

    mRNAs are transported, localized, and translated in axons of sensory neurons. However, little is known about the full repertoire of transcripts present in embryonic and adult sensory axons and how this pool of mRNAs dynamically changes during development. Here, we used a compartmentalized chamber to isolate mRNA from pure embryonic and adult sensory axons devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localized in sensory axons and that this repertoire changes during development toward adulthood. Embryonic axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are enriched in mRNAs encoding immune molecules with a role in nociception. Additionally, we show Tubulin-beta3 (Tubb3) mRNA is present only in embryonic axons, with Tubb3 locally synthesized in axons of embryonic, but not adult neurons where it is transported, thus validating our experimental approach. In summary, we provide the first complete catalog of embryonic and adult sensory axonal mRNAs. In addition we show that this pool of axonal mRNAs dynamically changes during development. These data provide an important resource for studies on the role of local protein synthesis in axon regeneration and nociception during neuronal development. PMID:21098654

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

    SciTech Connect

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

    1987-05-01

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

  10. The human and mouse SLC25A29 mitochondrial transporters rescue the deficient ornithine metabolism in fibroblasts of patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome.

    PubMed

    Camacho, José A; Rioseco-Camacho, Natalia

    2009-07-01

    The hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a disorder of the urea cycle (UCD) and ornithine degradation pathway caused by mutations in the mitochondrial ornithine transporter (ORNT1). Unlike other UCDs, HHH syndrome is characterized by a less severe and variable phenotype that we believe may, in part, be due to genes with redundant function to ORNT1, such as the previously characterized ORNT2 gene. We reasoned that SLC25A29, a member of the same subfamily of mitochondrial carrier proteins as ORNT1 and ORNT2, might also have overlapping function with ORNT1. Here, we report that both the human and mouse SLC25A29, previously identified as mitochondrial carnitine/acyl-carnitine transporter-like, when overexpressed transiently also rescues the impaired ornithine transport in cultured HHH fibroblasts. Moreover, we observed that, in the mouse, the Slc25a29 message is more significantly expressed in the CNS and cultured astrocytes when compared with the liver and kidney. These results suggest a potential physiologic role for the SLC25A29 transporter in the oxidation of fatty acids, ornithine degradation pathway, and possibly the urea cycle. Our results show that SLC25A29 is the third human mitochondrial ornithine transporter, designated as ORNT3, which may contribute to the milder and variable phenotype seen in patients with HHH syndrome. PMID:19287344

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

    PubMed

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

    2016-02-16

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

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

    PubMed Central

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

    2016-01-01

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

  13. Critical role of mitochondrial ROS is dependent on their site of production on the electron transport chain in ischemic heart

    PubMed Central

    Madungwe, Ngonidzashe B; Zilberstein, Netanel F; Feng, Yansheng; Bopassa, Jean C

    2016-01-01

    Abstract: Reactive oxygen species (ROS) generation has been implicated in many pathologies including ischemia/reperfusion (I/R) injury. This led to multiple studies on antioxidant therapies to treat cardiovascular diseases but paradoxically, results have so far been mixed as ROS production can be beneficial as a signaling mechanism and in cardiac protection via preconditioning interventions. We investigated whether the differential impact of increased ROS in injury as well as in protection could be explained by their site of production on the mitochondrial electron transport chain. Using amplex red to measure ROS production, we found that mitochondria isolated from hearts after I/R produced more ROS than non-ischemic when complex I substrate (glutamate/malate) was used. Interestingly, the substrates of complex II (succinate) and ubiquinone (sn-glycerol 3-phosphate, G3P) produced less ROS in mitochondria from I/R hearts compared to normal healthy hearts. The inhibitors of complex I (rotenone) and complex III (antimycin A) increased ROS production when glutamate/malate and G3P were used; in contrast, they reduced ROS production when the complex II substrate was used. Mitochondrial calcium retention capacity required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green fluorescence and was found to be higher when mitochondria were treated with G3P and succinate compared to glutamate/malate. Furthermore, Langendorff hearts treated with glutamate/malate exhibited reduced cardiac functional recovery and increased myocardial infarct size compared to hearts treated with G3P. Thus, ROS production by the stimulated respiratory chain complexes I and III has opposite roles: cardio-deleterious when produced in complex I and cardio-protective when produced in complex III. The mechanism of these ROS involves the inhibition of the mPTP opening, a key event in cell death following ischemia/reperfusion injury. PMID:27679744

  14. Critical role of mitochondrial ROS is dependent on their site of production on the electron transport chain in ischemic heart.

    PubMed

    Madungwe, Ngonidzashe B; Zilberstein, Netanel F; Feng, Yansheng; Bopassa, Jean C

    2016-01-01

    Reactive oxygen species (ROS) generation has been implicated in many pathologies including ischemia/reperfusion (I/R) injury. This led to multiple studies on antioxidant therapies to treat cardiovascular diseases but paradoxically, results have so far been mixed as ROS production can be beneficial as a signaling mechanism and in cardiac protection via preconditioning interventions. We investigated whether the differential impact of increased ROS in injury as well as in protection could be explained by their site of production on the mitochondrial electron transport chain. Using amplex red to measure ROS production, we found that mitochondria isolated from hearts after I/R produced more ROS than non-ischemic when complex I substrate (glutamate/malate) was used. Interestingly, the substrates of complex II (succinate) and ubiquinone (sn-glycerol 3-phosphate, G3P) produced less ROS in mitochondria from I/R hearts compared to normal healthy hearts. The inhibitors of complex I (rotenone) and complex III (antimycin A) increased ROS production when glutamate/malate and G3P were used; in contrast, they reduced ROS production when the complex II substrate was used. Mitochondrial calcium retention capacity required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green fluorescence and was found to be higher when mitochondria were treated with G3P and succinate compared to glutamate/malate. Furthermore, Langendorff hearts treated with glutamate/malate exhibited reduced cardiac functional recovery and increased myocardial infarct size compared to hearts treated with G3P. Thus, ROS production by the stimulated respiratory chain complexes I and III has opposite roles: cardio-deleterious when produced in complex I and cardio-protective when produced in complex III. The mechanism of these ROS involves the inhibition of the mPTP opening, a key event in cell death following ischemia/reperfusion injury. PMID:27679744

  15. Critical role of mitochondrial ROS is dependent on their site of production on the electron transport chain in ischemic heart

    PubMed Central

    Madungwe, Ngonidzashe B; Zilberstein, Netanel F; Feng, Yansheng; Bopassa, Jean C

    2016-01-01

    Abstract: Reactive oxygen species (ROS) generation has been implicated in many pathologies including ischemia/reperfusion (I/R) injury. This led to multiple studies on antioxidant therapies to treat cardiovascular diseases but paradoxically, results have so far been mixed as ROS production can be beneficial as a signaling mechanism and in cardiac protection via preconditioning interventions. We investigated whether the differential impact of increased ROS in injury as well as in protection could be explained by their site of production on the mitochondrial electron transport chain. Using amplex red to measure ROS production, we found that mitochondria isolated from hearts after I/R produced more ROS than non-ischemic when complex I substrate (glutamate/malate) was used. Interestingly, the substrates of complex II (succinate) and ubiquinone (sn-glycerol 3-phosphate, G3P) produced less ROS in mitochondria from I/R hearts compared to normal healthy hearts. The inhibitors of complex I (rotenone) and complex III (antimycin A) increased ROS production when glutamate/malate and G3P were used; in contrast, they reduced ROS production when the complex II substrate was used. Mitochondrial calcium retention capacity required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green fluorescence and was found to be higher when mitochondria were treated with G3P and succinate compared to glutamate/malate. Furthermore, Langendorff hearts treated with glutamate/malate exhibited reduced cardiac functional recovery and increased myocardial infarct size compared to hearts treated with G3P. Thus, ROS production by the stimulated respiratory chain complexes I and III has opposite roles: cardio-deleterious when produced in complex I and cardio-protective when produced in complex III. The mechanism of these ROS involves the inhibition of the mPTP opening, a key event in cell death following ischemia/reperfusion injury.

  16. The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters.

    PubMed

    Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

    2014-11-28

    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 inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.

  17. Brain-derived neurotrophic factor (BDNF)-induced mitochondrial motility arrest and presynaptic docking contribute to BDNF-enhanced synaptic transmission.

    PubMed

    Su, Bo; Ji, Yun-Song; Sun, Xu-lu; Liu, Xiang-Hua; Chen, Zhe-Yu

    2014-01-17

    Appropriate mitochondrial transport and distribution are essential for neurons because of the high energy and Ca(2+) buffering requirements at synapses. Brain-derived neurotrophic factor (BDNF) plays an essential role in regulating synaptic transmission and plasticity. However, whether and how BDNF can regulate mitochondrial transport and distribution are still unclear. Here, we find that in cultured hippocampal neurons, application of BDNF for 15 min decreased the percentage of moving mitochondria in axons, a process dependent on the activation of the TrkB receptor and its downstream PI3K and phospholipase-Cγ signaling pathways. Moreover, the BDNF-induced mitochondrial stopping requires the activation of transient receptor potential canonical 3 and 6 (TRPC3 and TRPC6) channels and elevated intracellular Ca(2+) levels. The Ca(2+) sensor Miro1 plays an important role in this process. Finally, the BDNF-induced mitochondrial stopping leads to the accumulation of more mitochondria at presynaptic sites. Mutant Miro1 lacking the ability to bind Ca(2+) prevents BDNF-induced mitochondrial presynaptic accumulation and synaptic transmission, suggesting that Miro1-mediated mitochondrial motility is involved in BDNF-induced mitochondrial presynaptic docking and neurotransmission. Together, these data suggest that mitochondrial transport and distribution play essential roles in BDNF-mediated synaptic transmission.

  18. The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters.

    PubMed

    Nancolas, Bethany; Guo, Lili; Zhou, Rong; Nath, Kavindra; Nelson, David S; Leeper, Dennis B; Blair, Ian A; Glickson, Jerry D; Halestrap, Andrew P

    2016-04-01

    Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitizing tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, whereas indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). In the present study, we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki2.5 μM) and co-operatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevisoocytes with K0.5 and Hill coefficient values of 36-40 μM and 1.65-1.85 respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50~ 7 μM) than other substrates including glutamate (IC50~ 20 μM). In isolated DB-1 melanoma cells 1-10 μM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 μM) decreased L-lactate output whereas increasing intracellular [L-lactate] > 5-fold, consistent with MCT inhibition. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND onL-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate (CHC). PMID:26831515

  19. The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters

    PubMed Central

    Nancolas, Bethany; Guo, Lili; Zhou, Rong; Nath, Kavindra; Nelson, David S.; Leeper, Dennis B.; Blair, Ian A.; Glickson, Jerry D.; Halestrap, Andrew P.

    2016-01-01

    Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitising tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, while indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). Here we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki 2.5 μM) and cooperatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 and Hill Coefficient values of 36–40 μM and 1.65–1.85. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50 ~7 μM) than other substrates including glutamate (IC50 ~20 μM). In isolated DB-1 melanoma cells 1–10 μM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 μM) decreased L-lactate output while increasing intracellular [L-lactate] > five-fold, consistent with MCT inhibition. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND on L-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate. PMID:26831515

  20. Axonal PPARγ promotes neuronal regeneration after injury.

    PubMed

    Lezana, Juan Pablo; Dagan, Shachar Y; Robinson, Ari; Goldstein, Ronald S; Fainzilber, Mike; Bronfman, Francisca C; Bronfman, Miguel

    2016-06-01

    PPARγ is a ligand-activated nuclear receptor best known for its involvement in adipogenesis and glucose homeostasis. PPARγ activity has also been associated with neuroprotection in different neurological disorders, but the mechanisms involved in PPARγ effects in the nervous system are still unknown. Here we describe a new functional role for PPARγ in neuronal responses to injury. We found both PPAR transcripts and protein within sensory axons and observed an increase in PPARγ protein levels after sciatic nerve crush. This was correlated with increased retrograde transport of PPARγ after injury, increased association of PPARγ with the molecular motor dynein, and increased nuclear accumulation of PPARγ in cell bodies of sensory neurons. Furthermore, PPARγ antagonists attenuated the response of sensory neurons to sciatic nerve injury, and inhibited axonal growth of both sensory and cortical neurons in culture. Thus, axonal PPARγ is involved in neuronal injury responses required for axonal regeneration. Since PPARγ is a major molecular target of the thiazolidinedione (TZD) class of drugs used in the treatment of type II diabetes, several pharmaceutical agents with acceptable safety profiles in humans are available. Our findings provide motivation and rationale for the evaluation of such agents for efficacy in central and peripheral nerve injuries. PMID:26446277

  1. Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons

    NASA Technical Reports Server (NTRS)

    Kirkpatrick, L. L.; Witt, A. S.; Payne, H. R.; Shine, H. D.; Brady, S. T.

    2001-01-01

    Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.

  2. Supercomplex assembly determines electron flux in the mitochondrial electron transport chain.

    PubMed

    Lapuente-Brun, Esther; Moreno-Loshuertos, Raquel; Acín-Pérez, Rebeca; Latorre-Pellicer, Ana; Colás, Carmen; Balsa, Eduardo; Perales-Clemente, Ester; Quirós, Pedro M; Calvo, Enrique; Rodríguez-Hernández, M A; Navas, Plácido; Cruz, Raquel; Carracedo, Ángel; López-Otín, Carlos; Pérez-Martos, Acisclo; Fernández-Silva, Patricio; Fernández-Vizarra, Erika; Enríquez, José Antonio

    2013-06-28

    The textbook description of mitochondrial respiratory complexes (RCs) views them as free-moving entities linked by the mobile carriers coenzyme Q (CoQ) and cytochrome c (cyt c). This model (known as the fluid model) is challenged by the proposal that all RCs except complex II can associate in supercomplexes (SCs). The proposed SCs are the respirasome (complexes I, III, and IV), complexes I and III, and complexes III and IV. The role of SCs is unclear, and their existence is debated. By genetic modulation of interactions between complexes I and III and III and IV, we show that these associations define dedicated CoQ and cyt c pools and that SC assembly is dynamic and organizes electron flux to optimize the use of available substrates.

  3. Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

    PubMed

    Yang, Chendong; Ko, Bookyung; Hensley, Christopher T; Jiang, Lei; Wasti, Ajla T; Kim, Jiyeon; Sudderth, Jessica; Calvaruso, Maria Antonietta; Lumata, Lloyd; Mitsche, Matthew; Rutter, Jared; Merritt, Matthew E; DeBerardinis, Ralph J

    2014-11-01

    Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

  4. Microfluidic control of axonal guidance

    NASA Astrophysics Data System (ADS)

    Gu, Ling; Black, Bryan; Ordonez, Simon; Mondal, Argha; Jain, Ankur; Mohanty, Samarendra

    2014-10-01

    The precision of axonal pathfinding and the accurate formation of functional neural circuitry are crucial for an organism during development as well as during adult central and peripheral nerve regeneration. While chemical cues are believed to be primarily responsible for axonal pathfinding, we hypothesize that forces due to localized fluid flow may directly affect neuronal guidance during early organ development. Here, we report direct evidence of fluid flow influencing axonal migration, producing turning angles of up to 90°. Microfluidic flow simulations indicate that an axon may experience significant bending force due to cross-flow, which may contribute to the observed axonal turning. This method of flow-based guidance was successfully used to fasciculate one advancing axon onto another, showcasing the potential of this technique to be used for the formation of in vitro neuronal circuits.

  5. Partial mitochondrial complex I inhibition induces oxidative damage and perturbs glutamate transport in primary retinal cultures. Relevance to Leber Hereditary Optic Neuropathy (LHON).

    PubMed

    Beretta, Simone; Wood, John P M; Derham, Barry; Sala, Gessica; Tremolizzo, Lucio; Ferrarese, Carlo; Osborne, Neville N

    2006-11-01

    Leber Hereditary Optic Neuropathy (LHON) is a maternally inherited form of visual loss, due to selective degeneration of retinal ganglion cells. Despite the established aetiological association between LHON and mitochondrial DNA mutations affecting complex I of the electron transport chain, the pathophysiology of this disorder remains obscure. Primary rat retinal cultures were exposed to increasing concentrations of rotenone to titrate complex I inhibition. Neural cells were more sensitive than Müller glial cells to rotenone toxicity. Rotenone induced an increase in mitochondrial-derived free radicals and lipid peroxidation. Sodium-dependent glutamate uptake, which is mostly mediated by the glutamate transporter GLAST expressed by Müller glial cells, was reduced dose-dependently by rotenone with no changes in GLAST expression. Our findings suggest that complex I-derived free radicals and disruption of glutamate transport might represent key elements for explaining the selective retinal ganglion cell death in LHON.

  6. Mitochondrial pathogenic mechanism and degradation in optineurin E50K mutation-mediated retinal ganglion cell degeneration

    PubMed Central

    Shim, Myoung Sup; Takihara, Yuji; Kim, Keun-Young; Iwata, Takeshi; Yue, Beatrice Y. J. T.; Inatani, Masaru; Weinreb, Robert N.; Perkins, Guy A.; Ju, Won-Kyu

    2016-01-01

    Mutations in optineurin (OPTN) are linked to the pathology of primary open angle glaucoma (POAG) and amyotrophic lateral sclerosis. Emerging evidence indicates that OPTN mutation is involved in accumulation of damaged mitochondria and defective mitophagy. Nevertheless, the role played by an OPTN E50K mutation in the pathogenic mitochondrial mechanism that underlies retinal ganglion cell (RGC) degeneration in POAG remains unknown. We show here that E50K expression induces mitochondrial fission-mediated mitochondrial degradation and mitophagy in the axons of the glial lamina of aged E50K−tg mice in vivo. While E50K activates the Bax pathway and oxidative stress, and triggers dynamics alteration-mediated mitochondrial degradation and mitophagy in RGC somas in vitro, it does not affect transport dynamics and fission of mitochondria in RGC axons in vitro. These results strongly suggest that E50K is associated with mitochondrial dysfunction in RGC degeneration in synergy with environmental factors such as aging and/or oxidative stress. PMID:27654856

  7. Cellular Strategies of Axonal Pathfinding

    PubMed Central

    Raper, Jonathan; Mason, Carol

    2010-01-01

    Axons follow highly stereotyped and reproducible trajectories to their targets. In this review we address the properties of the first pioneer neurons to grow in the developing nervous system and what has been learned over the past several decades about the extracellular and cell surface substrata on which axons grow. We then discuss the types of guidance cues and their receptors that influence axon extension, what determines where cues are expressed, and how axons respond to the cues they encounter in their environment. PMID:20591992

  8. Cellular strategies of axonal pathfinding.

    PubMed

    Raper, Jonathan; Mason, Carol

    2010-09-01

    Axons follow highly stereotyped and reproducible trajectories to their targets. In this review we address the properties of the first pioneer neurons to grow in the developing nervous system and what has been learned over the past several decades about the extracellular and cell surface substrata on which axons grow. We then discuss the types of guidance cues and their receptors that influence axon extension, what determines where cues are expressed, and how axons respond to the cues they encounter in their environment. PMID:20591992

  9. Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans

    PubMed Central

    Wojtovich, Andrew P.; Wei, Alicia Y.; Sherman, Teresa A.; Foster, Thomas H.; Nehrke, Keith

    2016-01-01

    Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the ‘singlet oxygen generator’ miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology. PMID:27440050

  10. Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans.

    PubMed

    Wojtovich, Andrew P; Wei, Alicia Y; Sherman, Teresa A; Foster, Thomas H; Nehrke, Keith

    2016-01-01

    Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the 'singlet oxygen generator' miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology. PMID:27440050

  11. In EXOG-depleted cardiomyocytes cell death is marked by a decreased mitochondrial reserve capacity of the electron transport chain.

    PubMed

    Tigchelaar, Wardit; De Jong, Anne Margreet; van Gilst, Wiek H; De Boer, Rudolf A; Silljé, Herman H W

    2016-07-01

    Depletion of mitochondrial endo/exonuclease G-like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease G-like-depleted cells, and this is marked by a decrease in mitochondrial reserve capacity. Neurohormonal stimulation with phenylephrine (PE) did not have an additive effect on the hypertrophic response induced by endo/exonuclease G-like depletion. Interestingly, PE-induced atrial natriuretic peptide (ANP) gene expression was completely abolished in endo/exonuclease G-like-depleted cells, suggesting a reverse signaling function of endo/exonuclease G-like. Endo/exonuclease G-like depletion initially resulted in increased mitochondrial OCR, but this declined upon PE stimulation. In particular, the reserve capacity of the mitochondrial respiratory chain and maximal respiration were the first indicators of perturbations in mitochondrial respiration, and these marked the subsequent decline in mitochondrial function. Although pathological stimulation accelerated these processes, prolonged EXOG depletion also resulted in a decline in mitochondrial function. At early stages of endo/exonuclease G-like depletion, mitochondrial ROS production was increased, but this did not affect mitochondrial DNA (mtDNA) integrity. After prolonged depletion, ROS levels returned to control values, despite hyperpolarization of the mitochondrial membrane. The mitochondrial dysfunction finally resulted in cell death, which appears to be mainly a form of necrosis. In conclusion, endo/exonuclease G-like plays an essential role in cardiomyocyte physiology. Loss of endo/exonuclease G-like results in diminished adaptation to pathological stress. The decline in maximal respiration and reserve capacity is the first sign of mitochondrial dysfunction that determines subsequent cell death. PMID:27417117

  12. Local Protein Synthesis in Axonal Growth Cones

    PubMed Central

    Šatkauskas, Saulius

    2007-01-01

    While initially thought to be essentially a developmental characteristic observed in artificial in vitro models, local protein synthesis in growth cones has been described in the adult, and more interestingly, during nerve regeneration. This emerging field is under intense investigation, revealing new functions of localized protein synthesis that include axon guidance, growth cone adaptation and sensitivity modulation at intermediate targets or axon regeneration. Here, we will review these functions and provide a short survey of the current knowledge on mechanisms of mRNA transport and regulation of localized protein synthesis. In addition, we will consider what lessons can be learned from localized protein synthesis in dendrites and what developments can be expected next in the field. This latter question relates to the crucial point of which technical strategy to adopt for an ideal and pertinent analysis of the phenomenon. PMID:19262143

  13. Tumor protein Tctp regulates axon development in the embryonic visual system

    PubMed Central

    Roque, Cláudio Gouveia; Wong, Hovy Ho-Wai; Lin, Julie Qiaojin; Holt, Christine E.

    2016-01-01

    The transcript encoding translationally controlled tumor protein (Tctp), a molecule associated with aggressive breast cancers, was identified among the most abundant in genome-wide screens of axons, suggesting that Tctp is important in neurons. Here, we tested the role of Tctp in retinal axon development in Xenopus laevis. We report that Tctp deficiency results in stunted and splayed retinotectal projections that fail to innervate the optic tectum at the normal developmental time owing to impaired axon extension. Tctp-deficient axons exhibit defects associated with mitochondrial dysfunction and we show that Tctp interacts in the axonal compartment with myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl2 family. Mcl1 knockdown gives rise to similar axon misprojection phenotypes, and we provide evidence that the anti-apoptotic activity of Tctp is necessary for the normal development of the retinotectal projection. These findings suggest that Tctp supports the development of the retinotectal projection via its regulation of pro-survival signalling and axonal mitochondrial homeostasis, and establish a novel and fundamental role for Tctp in vertebrate neural circuitry assembly. PMID:26903505

  14. The mitochondrial ornithine transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms.

    PubMed

    Fiermonte, Giuseppe; Dolce, Vincenza; David, Laura; Santorelli, Filippo Maria; Dionisi-Vici, Carlo; Palmieri, Ferdinando; Walker, John E

    2003-08-29

    Two isoforms of the human ornithine carrier, ORC1 and ORC2, have been identified by overexpression of the proteins in bacteria and by study of the transport properties of the purified proteins reconstituted into liposomes. Both transport L-isomers of ornithine, lysine, arginine, and citrulline by exchange and by unidirectional mechanisms, and they are inactivated by the same inhibitors. ORC2 has a broader specificity than ORC1, and L- and D-histidine, L-homoarginine, and D-isomers of ornithine, lysine, and ornithine are all substrates. Both proteins are expressed in a wide range of human tissues, but ORC1 is the predominant form. The highest levels of expression of both isoforms are in the liver. Five mutant forms of ORC1 associated with the human disease hyperornithinemia-hyperammonemia-homocitrullinuria were also made. The mutations abolish the transport properties of the protein. In patients with hyperornithinemia-hyperammonemia-homocitrullinuria, isoform ORC2 is unmodified, and its presence compensates partially for defective ORC1. PMID:12807890

  15. Axon guidance: FLRTing promotes attraction.

    PubMed

    Lowery, Laura Anne

    2014-03-01

    A recent study demonstrates a new mechanism by which crosstalk between multiple guidance cues is integrated during axon pathfinding. FLRT3 is a novel co-receptor for Robo1 that acts as a context-dependent modulator of Netrin-1 attraction in thalamocortical axons.

  16. Reduction in cardiolipin decreases mitochondrial spare respiratory capacity and increases glucose transport into and across human brain cerebral microvascular endothelial cells.

    PubMed

    Nguyen, Hieu M; Mejia, Edgard M; Chang, Wenguang; Wang, Ying; Watson, Emily; On, Ngoc; Miller, Donald W; Hatch, Grant M

    2016-10-01

    Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin is a mitochondrial phospholipid required for function of the electron transport chain and ATP generation. We examined the role of cardiolipin in maintaining mitochondrial function necessary to support barrier properties of brain microvessel endothelial cells. Knockdown of the terminal enzyme of cardiolipin synthesis, cardiolipin synthase, in hCMEC/D3 cells resulted in decreased cellular cardiolipin levels compared to controls. The reduction in cardiolipin resulted in decreased mitochondrial spare respiratory capacity, increased pyruvate kinase activity, and increased 2-deoxy-[(3) H]glucose uptake and glucose transporter-1 expression and localization to membranes in hCMEC/D3 cells compared to controls. The mechanism for the increase in glucose uptake was an increase in adenosine-5'-monophosphate kinase and protein kinase B activity and decreased glycogen synthase kinase 3 beta activity. Knockdown of cardiolipin synthase did not affect permeability of fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell(®) inserts. In contrast, knockdown of cardiolipin synthase resulted in an increase in 2-deoxy-[(3) H]glucose transport across these monolayers compared to controls. The data indicate that in hCMEC/D3 cells, spare respiratory capacity is dependent on cardiolipin. In addition, reduction in cardiolipin in these cells alters their cellular energy status and this results in increased glucose transport into and across hCMEC/D3 monolayers. Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. In human adult brain endothelial cell hCMEC/D3 monolayers cultured on Transwell(®) plates, knockdown of cardiolipin synthase results in decrease in mitochondrial

  17. Signaling from Axon Guidance Receptors

    PubMed Central

    Bashaw, Greg J.; Klein, Rüdiger

    2010-01-01

    Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues—netrins, semaphorins, ephrins, and slits—have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance. PMID:20452961

  18. Signaling from axon guidance receptors.

    PubMed

    Bashaw, Greg J; Klein, Rüdiger

    2010-05-01

    Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues--netrins, semaphorins, ephrins, and slits--have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance. PMID:20452961

  19. The involvement of mitochondrial phosphate transporter in accelerating bud dormancy release during chilling treatment of tree peony (Paeonia suffruticosa).

    PubMed

    Huang, Xin; Zhu, Wei; Dai, Silan; Gai, Shupeng; Zheng, Guosheng; Zheng, Chengchao

    2008-09-01

    A cDNA clone was isolated from tree peony (Paeonia suffruticosa) subtractive cDNA library of burst buds and characterized with regard to its sequence, expression in response to chilling treatment during the release of bud dormancy, and its function in transgenic Arabidopsis thaliana. The clone, designated as PsMPT, contains 1,615 nucleotides with an open reading frame of 1,119 nucleotides, and the deduced amino acid sequence shows high homology with mitochondrial phosphate transporters (MPTs) from various organisms. The mRNA accumulation of PsMPT in tree peony was strongly induced by chilling treatment during the release of bud dormancy. When the treated plants were transferred to normal growth conditions, the level of PsMPT transcripts induced by sufficient chilling could be maintained high, whereas that induced by insufficient chilling decreased sharply. The transgenic Arabidopsis plants that overexpress PsMPT showed rapid growth and earlier flowering than wild-type plants. ATP contents in the transgenic plants were much higher than that in wild-type plants through various developmental stages. Together, these results suggest that the product of PsMPT is a MPT and might play an important role during the release of bud dormancy in tree peony.

  20. [F-18]Fluorodihydrorotenone: Synthesis and evaluation of a mitochondrial electron transport chain (ETC) complex I probe for PET

    SciTech Connect

    VanBrocklin, H.F.; Enas, J.D.; Hanrahan, S.M.

    1994-05-01

    The mitochondrial electron transport chain (ETC) consists of five enzyme complexes (I-V) which participate in the transfer of electrons to oxygen and phosphorylation of ADP (oxidative phosphorylation). ETC dysfunction has been linked to several genetic neurological diseases as well as implicated in Parkinson`s (complex I) and Huntington`s (complex I) disease and normal aging processes. Dihydrorotenone (DHR) is a specific high affinity inhibitor of complex I. In order to develop a PET tracer for complex I, we have labeled DHR with fluorine-18. The tosylate precursor was produced in three steps from commercially available rotenone. Fluorine-18 was introduced by nucleophilic displacement of the tosylate using tetrabutyl-ammonium fluoride. Subsequent oxidation with MnO{sub 2} and HPLC purification gave the desired [{sup 18}F]fluoro-DHR. Initial biodistribution studies were carried out in {approximately}200 g male Sprague-Dawley rats. The tracer was taken up rapidly in the heart, an organ highly enriched with mitochondria, (5.5-6% injected dose (ID)/g at 30 minutes) and in the brain ({approximately}1.5% ID/g at 1 hour).

  1. The intricate relationship between microtubules and their associated motor proteins during axon growth and maintenance

    PubMed Central

    2013-01-01

    The hallmarks of neurons are their slender axons which represent the longest cellular processes of animals and which act as the cables that electrically wire the brain, and the brain to the body. Axons extend along reproducible paths during development and regeneration, and they have to be maintained for the lifetime of an organism. Both axon extension and maintenance essentially depend on the microtubule (MT) cytoskeleton. For this, MTs organize into parallel bundles that are established through extension at the leading axon tips within growth cones, and these bundles then form the architectural backbones, as well as the highways for axonal transport essential for supply and intracellular communication. Axon transport over these enormous distances takes days or even weeks and is a substantial logistical challenge. It is performed by kinesins and dynein/dynactin, which are molecular motors that form close functional links to the MTs they walk along. The intricate machinery which regulates MT dynamics, axonal transport and the motors is essential for nervous system development and function, and its investigation has huge potential to bring urgently required progress in understanding the causes of many developmental and degenerative brain disorders. During the last years new explanations for the highly specific properties of axonal MTs and for their close functional links to motor proteins have emerged, and it has become increasingly clear that motors play active roles also in regulating axonal MT networks. Here, I will provide an overview of these new developments. PMID:24010872

  2. Evidence that multiple species of aminoacylated transfer RNA are present in regenerating optic axons of goldfish

    SciTech Connect

    Zanakis, M.F.; Eskin, B.; Ingoglia, N.A.

    1984-02-01

    This study reports that 4S RNA present in regenerating optic axons of goldfish is likely to be transfer RNA. Evidence is also presented which indicates that this transfer RNA is similar to transfer RNA found in tectal cells and that its aminoacylation is likely to occur both in retinal ganglion cells prior to axonal transport as well as in the axon itself. Fish with regenerating optic nerves received intraocular injections of (/sup 3/H)uridine followed 4 days later by intracranial injections of (/sup 14/C)uridine. Radioactive tectal 4S RNA was isolated 6 days after (/sup 3/H)uridine injections and chromatographed by BD cellulose chromatography. Optical density as well as radioactivity profiles for both (/sup 14/C)4S RNA (from tectal cells) and (/sup 3/H)4S RNA (90% of which originated from regenerating optic axons) were found to be similar to E. coli transfer RNA optical density profiles, indicating that the intra-axonal 4S RNA is likely to be transfer RNA. Moreover, comparisons of /sup 3/H//sup 14/C suggest that intra-axonal and cellular 4S RNAs are composed of similar species of transfer RNA. Results of other experiments indicate that aminoacylation of axonally transported tRNA occurs both in the retina and in optic axons subsequent to axonal transport.

  3. The zinc finger RNA binding protein, ZFR, contributes to axon guidance in Caenorhabditis elegans.

    PubMed

    Kjærgaard, Tine; Desdorf, Rasmus; Heuck, Anders; Olsen, Anders; Lykke-Hartmann, Karin

    2015-02-15

    ZFR is an ancient and highly conserved chromosome-associated protein from nematodes to mammals, embryologically expressed in most species, with the exception of the nematode Caenorhabditis elegans. The ZFR encodes zinc and RNA binding protein, and in rat, the nuclear-cytoplasmic shuttling ZFR has been found with transport and translation-associated RNA granule-like structures in the somatodendritic compartments of hippocampal neurons. The majority of axons cross the midline before projecting to their contralateral synaptic target and this crossing decision is under tight control. Molecular factors contributing to these processes have been identified, although the mechanisms are not fully understood. In this study, we tested the role of ceZFR in axon guidance using ceZfr RNAi-treated animals to analyse axon midline crossing, axon fasciculation and cord commissures. In adult stages, RNAi-induced depletion of the ceZfr transcript leads to several phenotypes related to axon guidance. A midline crossing defect was observed in the ventral nerve cord (VNC) in axon type D, DD/VD motoneuron axons and axon type 1, interneuron axons. We further detected a dorsal nerve cord (DNC) axon fasciculation. Some ceZfr RNAi-treated animals revealed that cord commissures fail to reach their synaptic target. We provide evidence that ceZFR has a role in axon guidance. When Zfr was depleted by RNAi, the phenotypes are characterized by defects in axon midline crossing, axon defasciculation and cord commissures. Our results thus support the hypothesis that ZFR has essential roles during neurogenesis, and could support early steps of RNA transport and localization through RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling.

  4. Increased Axonal Ribosome Numbers Is an Early Event in the Pathogenesis of Amyotrophic Lateral Sclerosis

    PubMed Central

    Verheijen, Mark H. G.; Peviani, Marco; Hendricusdottir, Rita; Bell, Erin M.; Lammens, Martin; Smit, August B.; Bendotti, Caterina; van Minnen, Jan

    2014-01-01

    Myelinating glia cells support axon survival and functions through mechanisms independent of myelination, and their dysfunction leads to axonal degeneration in several diseases. In amyotrophic lateral sclerosis (ALS), spinal motor neurons undergo retrograde degeneration, and slowing of axonal transport is an early event that in ALS mutant mice occurs well before motor neuron degeneration. Interestingly, in familial forms of ALS, Schwann cells have been proposed to slow disease progression. We demonstrated previously that Schwann cells transfer polyribosomes to diseased and regenerating axons, a possible rescue mechanism for disease-induced reductions in axonal proteins. Here, we investigated whether elevated levels of axonal ribosomes are also found in ALS, by analysis of a superoxide dismutase 1 (SOD1)G93A mouse model for human familial ALS and a patient suffering from sporadic ALS. In both cases, we found that the disorder was associated with an increase in the population of axonal ribosomes in myelinated axons. Importantly, in SOD1G93A mice, the appearance of axonal ribosomes preceded the manifestation of behavioral symptoms, indicating that upregulation of axonal ribosomes occurs early in the pathogenesis of ALS. In line with our previous studies, electron microscopy analysis showed that Schwann cells might serve as a source of axonal ribosomes in the disease-compromised axons. The early appearance of axonal ribosomes indicates an involvement of Schwann cells early in ALS neuropathology, and may serve as an early marker for disease-affected axons, not only in ALS, but also for other central and peripheral neurodegenerative disorders. PMID:24498056

  5. Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

    PubMed Central

    Kucejova, Blanka; Li, Li; Wang, Xiaowen; Giannattasio, Sergio; Chen, Xin Jie

    2009-01-01

    In Saccharomyces cerevisiae, SAL1 encodes a Ca2+-binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Δ exacerbates the respiratory deficiency and mtDNA instability of ggc1Δ, shy1Δ and mtg1Δ mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+-binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria. PMID:18431598

  6. Optic nerve degeneration and mitochondrial dysfunction: genetic and acquired optic neuropathies.

    PubMed

    Carelli, Valerio; Ross-Cisneros, Fred N; Sadun, Alfredo A

    2002-05-01

    Selective degeneration of the smallest fibers (papillo-macular bundle) of the human optic nerve occurs in a large number of optic neuropathies characterized primarily by loss of central vision. The pathophysiology that underlies this peculiar pattern of cell involvement probably reflects different forms of genetic and acquired mitochondrial dysfunction. Maternally inherited Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (Kjer disease), the optic atrophy of Leigh's syndrome, Friedreich ataxia and a variety of other conditions are examples of inherited mitochondrial disorders with different etiologies. Tobacco-alcohol amblyopia (TAA), the Cuban epidemic of optic neuropathy (CEON) and other dietary (Vitamins B, folate deficiencies) optic neuropathies, as well as toxic optic neuropathies such as due to chloramphenicol, ethambutol, or more rarely to carbon monoxide, methanol and cyanide are probably all related forms of acquired mitochondrial dysfunction. Biochemical and cellular studies in LHON point to a partial defect of respiratory chain function that may generate either an ATP synthesis defect and/or a chronic increase of oxidative stress. Histopathological studies in LHON cases and a rat model mimicking CEON revealed a selective loss of retinal ganglion cells (RGCs) and the corresponding axons, particularly in the temporal-central part of the optic nerve. Anatomical peculiarities of optic nerve axons, such as the asymmetric pattern of myelination, may have functional implications on energy dependence and distribution of mitochondrial populations in the different sections of the nerve. Histological evidence suggests impaired axonal transport of mitochondria in LHON and in the CEON-like rat model, indicating a possible common pathophysiology for this category of optic neuropathies. Histological evidence of myelin pathology in LHON also suggests a role for oxidative stress, possibly affecting the oligodendrocytes of the optic nerves.

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

    PubMed Central

    White, Robin; Krämer-Albers, Eva-Maria

    2014-01-01

    In vertebrate nervous systems myelination of neuronal axons has evolved to increase conduction velocity of electrical impulses with minimal space and energy requirements. Myelin is formed by specialized glial cells which ensheath axons with a lipid-rich insulating membrane. Myelination is a multi-step process initiated by axon-glia recognition triggering glial polarization followed by targeted myelin membrane expansion and compaction. Thereby, a myelin sheath of complex subdomain structure is established. Continuous communication between neurons and glial cells is essential for myelin maintenance and axonal integrity. A diverse group of diseases, from multiple sclerosis to schizophrenia, have been linked to malfunction of myelinating cells reflecting the physiological importance of the axon-glial unit. This review describes the mechanisms of axonal signal integration by oligodendrocytes emphasizing the central role of the Src-family kinase Fyn during central nervous system (CNS) myelination. Furthermore, we discuss myelin membrane trafficking with particular focus on endocytic recycling and the control of proteolipid protein (PLP) transport by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Finally, PLP mistrafficking is considered in the context of myelin diseases. PMID:24431989

  8. Cytoplasmic mechanisms of axonal and dendritic growth in neurons.

    PubMed

    Heidemann, S R

    1996-01-01

    The structural mechanisms responsible for the gradual elaboration of the cytoplasmic elongation of neurons are reviewed. In addition to discussing recent work, important older work is included to inform newcomers to the field how the current perspective arose. The highly specialized axon and the less exaggerated dendrite both result from the advance of the motile growth cone. In the area of physiology, studies in the last decade have directly confirmed the classic model of the growth cone pulling forward and the axon elongating from this tension. Particularly in the case of the axon, cytoplasmic elongation is closely linked to the formation of an axial microtubule bundle from behind the advancing growth cone. Substantial progress has been made in understanding the expression of microtubule-associated proteins during neuronal differentiation to stiffen and stabilize axonal microtubules, providing specialized structural support. Studies of membrane organelle transport along the axonal microtubules produced an explosion of knowledge about ATPase molecules serving as motors driving material along microtubule rails. However, most aspects of the cytoplasmic mechanisms responsible for neurogenesis remain poorly understood. There is little agreement on mechanisms for the addition of new plasma membrane or the addition of new cytoskeletal filaments in the growing axon. Also poorly understood are the mechanisms that couple the promiscuous motility of the growth cone to the addition of cytoplasmic elements.

  9. Abnormal morphology of myelin and axon pathology in murine models of multiple sclerosis.

    PubMed

    Bando, Yoshio; Nomura, Taichi; Bochimoto, Hiroki; Murakami, Koichi; Tanaka, Tatsuhide; Watanabe, Tsuyoshi; Yoshida, Shigetaka

    2015-02-01

    Demyelination and axonal damage are responsible for neurological deficits in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. However, the pathology of axonal damage in MS is not fully understood. In this study, histological analysis of morphological changes of axonal organelles during demyelination in murine models was investigated by scanning electron microscopy (SEM) using an osmium-maceration method. In cuprizone-induced demyelination, SEM showed typical morphology of demyelination in the corpus callosum of mouse brain. In contrast, SEM displayed variations in ultrastructural abnormalities of myelin structures and axonal organelles in spinal cord white matter of experimental autoimmune encephalomyelitis (EAE) mice, an animal model of MS. Myelin detachment and excessive myelin formation were observed as typical morphological myelin abnormalities in EAE. In addition, well-developed axoplasmic reticulum-like structures and accumulated mitochondria were observed in tortuous degenerating/degenerated axons and the length of mitochondria in axons of EAE spinal cord was shorter compared with naïve spinal cord. Immunohistochemistry also revealed dysfunction of mitochondrial fusion/fission machinery in EAE spinal cord axons. Moreover, the number of Y-shaped mitochondria was significantly increased in axons of the EAE spinal cord. Axonal morphologies in myelin basic protein-deficient shiverer mice were similar to those in EAE. However, shiverer mice had "tortuous" (S-curve shaped mitochondria) and larger mitochondria compared with wild-type and EAE mice. Lastly, analysis of human MS patient autopsied brains also demonstrated abnormal myelin structures in demyelinating lesions. These results indicate that morphological abnormalities of myelin and axonal organelles play important role on the pathogenesis of axonal injury in demyelinating diseases.

  10. Differential compartmentalization of mRNAs in squid giant axon.

    PubMed

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

    1996-11-01

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

  11. Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons

    PubMed Central

    Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E.

    2015-01-01

    The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration. PMID:26366578

  12. Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons.

    PubMed

    Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E

    2015-01-01

    The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.

  13. Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon

    PubMed Central

    Ma, Marek

    2013-01-01

    Axonal injury and degeneration, whether primary or secondary, contribute to the morbidity and mortality seen in many acquired and inherited central nervous system (CNS) and peripheral nervous system (PNS) disorders, such as traumatic brain injury, spinal cord injury, cerebral ischemia, neurodegenerative diseases, and peripheral neuropathies. The calpain family of proteases has been mechanistically linked to the dysfunction and degeneration of axons. While the direct mechanisms by which transection, mechanical strain, ischemia, or complement activation trigger intra-axonal calpain activity are likely different, the downstream effects of unregulated calpain activity may be similar in seemingly disparate diseases. In this review, a brief examination of axonal structure is followed by a focused overview of the calpain family. Finally, the mechanisms by which calpains may disrupt the axonal cytoskeleton, transport, and specialized domains (axon initial segment, nodes, and terminals) are discussed. PMID:23969238

  14. The K+/site and H+/site stoichiometry of mitochondrial electron transport.

    PubMed

    Reynafarje, B; Lehninger, A L

    1978-09-25

    Electrode measurements of the average number of H+ ejected and K+ taken up (in the presence of valinomycin) per pair of electrons passing the energy-conserving sites of the respiratory chain of rat liver and rat heart mitochondria have given identical values of the H+/site and 5+/site ratios very close to 4 in the presence of N-ethylmaleimide, an inhibitor of interfering respiration-coupled uptake of H+ + H2PO4-. The K+/site uptake ratio of 4 not only shows that inward movement of K+ provides quantitative charge-compensation for the 4 H+ ejected, but also confirms that 4 charges are separated per pair of electrons per site. When N-ethylmaleimide is omitted, the H+/site ejection ratio is depressed, because of the interfering secondary uptake of H/+ with H2PO4- on the phosphate carrier, but the K+/site uptake ratio remains at 4.0. Addition of phosphate or acetate, which can carry H+ into respiring mitochondria, further depresses the H+/site ratio, but does not affect the K+/site ratio, which remains at 4.0. These and other considerations thus confirm our earlier stoichiometric measurements that the average H+/site ratio is 4.0 and also show that the K+/site uptake ratio can be used as a measure of the intrinsic H+/site ratio, regardless of the presence of phosphate in the medium and without the necessity of adding N-ethylmaleimide or other inhibitors of H+ + H2PO4- transport.

  15. Phosphatidylinositol 3-kinase and the actin network are not required for the stimulation of glucose transport caused by mitochondrial uncoupling: comparison with insulin action.

    PubMed Central

    Tsakiridis, T; Vranic, M; Klip, A

    1995-01-01

    In L6 myotubes insulin stimulates glucose transport through the translocation of glucose transporters GLUT1, GLUT3 and GLUT4 from intracellular stores to the plasma membrane. An intact actin network and phosphatidylinositol 3-kinase activity are required for this process. Glucose transport is also stimulated by the mitochondrial ATP-production uncoupler dinitrophenol. We show here that, in serum-depleted myotubes, dinitrophenol induced translocation of GLUT1 and GLUT4, but not GLUT3. This response was not affected by inhibiting phosphatidylinositol 3-kinase or disassembling the actin network. Insulin, but not dinitrophenol, caused tyrosine phosphorylation of several polypeptides, including the insulin-receptor substrate-1 and mitogen-activated protein kinase. Similarly, insulin, but not dinitrophenol, caused actin reorganization, which was inhibited by wortmannin. We conclude that insulin and dinitrophenol stimulate glucose transport by different mechanisms. Images Figure 2 Figure 3 Figure 4 PMID:7619042

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

    PubMed

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

    2016-01-01

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

  17. No evidence for chronic demyelination in spared axons after spinal cord injury in a mouse.

    PubMed

    Lasiene, Jurate; Shupe, Larry; Perlmutter, Steve; Horner, Philip

    2008-04-01

    The pattern of remyelination after traumatic spinal cord injury remains elusive, with animal and human studies reporting partial to complete demyelination followed by incomplete remyelination. In the present study, we found that spared rubrospinal tract (RST) axons of passage traced with actively transported dextrans and examined caudally to the lesion 12 weeks after mouse spinal cord contusion injury were fully remyelinated. Spared axons exhibited a marginally reduced myelin thickness and significantly shorter internodes. CASPR (contactin-associated protein) and K(v)1.2 channels were used to identify internodes and paranodal protein distribution properties were used as an index of myelin integrity. This is the first time the CNS myelin internode length was measured in a mouse. To better understand the significance of shortened internodes and thinner myelin in spared axons, we modeled conduction properties using McIntyre's et al. model of myelinated axons. Mathematical modeling predicted a 21% decrease in the conduction velocity of remyelinated RST axons attributable to shortened internodes. To determine whether demyelination could be present on axons exhibiting a pathological transport system, we used the retroviral reporter system. Virally delivered green fluorescent protein unveiled a small population of dystrophic RST axons that persist chronically with evident demyelination or abnormal remyelination. Collectively, these data show that lasting demyelination in spared axons is rare and that remyelination of axons of passage occurs in the chronically injured mouse spinal cord. PMID:18400887

  18. Flexible change and cooperation between mitochondrial electron transport and cytosolic glycolysis as the basis for chilling tolerance in tomato plants.

    PubMed

    Shi, Kai; Fu, Li-Jun; Zhang, Shuai; Li, Xin; Liao, Yang-Wen-Ke; Xia, Xiao-Jian; Zhou, Yan-Hong; Wang, Rong-Qing; Chen, Zhi-Xiang; Yu, Jing-Quan

    2013-02-01

    To find if cytosolic glycolysis dynamical metabolism plays a role in mediating respiration homeostasis and its relationship with mitochondrial electron transport chain (miETC) flexibility, we selected two tomato genotypes that differ in chilling tolerance and compared the responses of miETC, cytosolic glycolysis and respiratory homeostasis at 7 °C. Our results showed that the transcripts of both classical and bypass component genes for miETC and glycolysis were comparable for both genotypes when grown at 25 °C. However, there was a rapid global increase in the expression of most respiratory genes in response to chilling at 7 °C for both genotypes. When normally grown plant was set as the control for each genotype, the transcripts of most COX family members, ATP synthase, AOX1b, and UCP are highly up-regulated in chilling-tolerant Zhefen No. 208 plants in contrast to the sensitive Zhefen No. 212 plants. Both genotypes mobilized the energy-saving sucrose synthase pathway for sucrose degradation by cytosolic glycolysis, but this mechanism is evidently more effective in tolerant Zhefen No. 208 plants. Furthermore, only Zhefen No. 208 plants were able to partially switch from low-energy efficiency pathways to ATP conserving pathways to carry out fructose-6-phosphate conversion and pyruvate production. This metabolic flexibility in miETC and cytosolic glycolysis were coupled to higher ATP synthesis and lower ROS accumulation, which may be essential for sustaining the higher leaf respiration and homeostasis of chilling-tolerant plants.

  19. On the location of the H+-extruding steps in site 2 of the mitochondrial electron transport chain.

    PubMed

    Alexandre, A; Galiazzo, F; Lehninger, A L

    1980-11-25

    The location of the H+-translocating reactions within energy-conserving Site 2 of the mitochondrial electron transport chain was evaluated from two sets of data. In the first, the H+/2e- ejection ratios and Ca2+/2e- uptake ratios were compared for electron flow from succinate dehydrogenase, whose active site is on the matrix side of the inner membrane and from glycerol phosphate dehydrogenase, whose active site is on the cytosolic side. In intact rat liver mitochondria both substrates yielded H+/2e- ejection ratios close to 4.0 and Ca2+/2e- uptake ratios close to 1.0 during antimycin-sensitive reduction of ferricyanide. With rat liver mitoplasts and ferricytochrome c as electron acceptor, both substrates again gave the same stoichiometric ratios. The second approach involved determination of the sidedness of H+ formation during electron flow from succinate to ferricyanide via bypass of the antimycin block of the cytochrome b.c1 complex provided by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), under conditions in which the TMPD-TMPD+ couple does not act as a membrane-penetrating protonophore. Electron flow in this system was inhibited by 2-then-oyltrifluoroacetone, indicating that TMPD probably accepts electrons from ubiquinol. The 2 H+ formed in this system were not delivered into the matrix but appeared directly in the medium in the absence of a protonophore. To accommodate the available evidence on Site 2 substrates, it is concluded that the substrate hydrogens are first transferred to ubiquinone, 2 H+ per 2e then appear in the medium by protolytic dehydrogenation of a species of ubiquinol or ubiquinol-protein having the appropriate sidedness (designated Site 2A), and the other 2 H+ are translocated from the matrix to the medium on passage of 2e- through the cytochrome b x c1 complex (designated Site 2B).

  20. Mitochondrial optic neuropathies – Disease mechanisms and therapeutic strategies

    PubMed Central

    Yu-Wai-Man, Patrick; Griffiths, Philip G.; Chinnery, Patrick F.

    2011-01-01

    Leber hereditary optic neuropathy (LHON) and autosomal-dominant optic atrophy (DOA) are the two most common inherited optic neuropathies in the general population. Both disorders share striking pathological similarities, marked by the selective loss of retinal ganglion cells (RGCs) and the early involvement of the papillomacular bundle. Three mitochondrial DNA (mtDNA) point mutations; m.3460G>A, m.11778G>A, and m.14484T>C account for over 90% of LHON cases, and in DOA, the majority of affected families harbour mutations in the OPA1 gene, which codes for a mitochondrial inner membrane protein. Optic nerve degeneration in LHON and DOA is therefore due to disturbed mitochondrial function and a predominantly complex I respiratory chain defect has been identified using both in vitro and in vivo biochemical assays. However, the trigger for RGC loss is much more complex than a simple bioenergetic crisis and other important disease mechanisms have emerged relating to mitochondrial network dynamics, mtDNA maintenance, axonal transport, and the involvement of the cytoskeleton in maintaining a differential mitochondrial gradient at sites such as the lamina cribosa. The downstream consequences of these mitochondrial disturbances are likely to be influenced by the local cellular milieu. The vulnerability of RGCs in LHON and DOA could derive not only from tissue-specific, genetically-determined biological factors, but also from an increased susceptibility to exogenous influences such as light exposure, smoking, and pharmacological agents with putative mitochondrial toxic effects. Our concept of inherited mitochondrial optic neuropathies has evolved over the past decade, with the observation that patients with LHON and DOA can manifest a much broader phenotypic spectrum than pure optic nerve involvement. Interestingly, these phenotypes are sometimes clinically indistinguishable from other neurodegenerative disorders such as Charcot-Marie-Tooth disease, hereditary spastic

  1. Calcium-dependent proteasome activation is required for axonal neurofilament degradation.

    PubMed

    Park, Joo Youn; Jang, So Young; Shin, Yoon Kyung; Suh, Duk Joon; Park, Hwan Tae

    2013-12-25

    Even though many studies have identified roles of proteasomes in axonal degeneration, the molecular mechanisms by which axonal injury regulates proteasome activity are still unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regulator of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were significantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swelling, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wallerian degeneration.

  2. Axonal localization of neuritin/CPG15 mRNA in neuronal populations through distinct 5' and 3' UTR elements.

    PubMed

    Merianda, Tanuja T; Gomes, Cynthia; Yoo, Soonmoon; Vuppalanchi, Deepika; Twiss, Jeffery L

    2013-08-21

    Many neuronal mRNAs are actively transported into distal axons. The 3' untranslated regions (UTRs) of axonal mRNAs often contain cues for their localization. The 3' UTR of neuritin mRNA was shown to be sufficient for localization into axons of hippocampal neurons. Here, we show that neuritin mRNA localizes into axons of rat sensory neurons, but this is predominantly driven by the 5' rather than 3' UTR. Neuritin mRNA shifts from cell body to axon predominantly after nerve crush injury, suggesting that it encodes a growth-associated protein. Consistent with this, overexpression of neuritin increases axon growth but only when its mRNA localizes into the axons. PMID:23966695

  3. Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points

    PubMed Central

    Pittman, Andrew J.; Law, Mei-Yee; Chien, Chi-Bin

    2008-01-01

    Summary Navigating axons respond to environmental guidance signals, but can also follow axons that have gone before—pioneer axons. Pioneers have been studied extensively in simple systems, but the role of axon-axon interactions remains largely unexplored in large vertebrate axon tracts, where cohorts of identical axons could potentially use isotypic interactions to guide each other through multiple choice points. Furthermore, the relative importance of axon-axon interactions compared to axon-autonomous receptor function has not been assessed. Here we test the role of axon-axon interactions in retinotectal development, by devising a technique to selectively remove or replace early-born retinal ganglion cells (RGCs). We find that early RGCs are both necessary and sufficient for later axons to exit the eye. Furthermore, introducing misrouted axons by transplantation reveals that guidance from eye to tectum relies heavily on interactions between axons, including both pioneer-follower and community effects. We conclude that axon-axon interactions and ligand-receptor signaling have coequal roles, cooperating to ensure the fidelity of axon guidance in developing vertebrate tracts. PMID:18653554

  4. Identification of precursor microRNAs within distal axons of sensory neuron

    PubMed Central

    Kim, Hak Hee; Kim, Paul; Phay, Monichan; Yoo, Soonmoon

    2015-01-01

    A set of specific precursor microRNAs (pre-miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre-miRNAs are also transported into distal axons to autonomously regulate intra-axonal protein synthesis. Here, we show that a subset of pre-miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre-miRNAs (let 7c-a and pre-miRs-16, 23a, 25, 125b-1, 433, and 541) showed elevated axonal levels, while others (pre-miRs-138-2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre-miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons. PMID:25919946

  5. Inhibition of dynein but not kinesin induces aberrant focal accumulation of neurofilaments within axonal neurites.

    PubMed

    Motil, Jennifer; Dubey, Maya; Chan, Walter K-H; Shea, Thomas B

    2007-08-20

    Studies from several laboratories indicate that the microtubule motors kinesin and dynein respectively participate in anterograde and retrograde axonal transport of neurofilaments. Inhibition of dynein function by transfection with a construct expressing dynamitin or intracellular delivery of anti-dynein antibodies accelerates anterograde transport, which has been interpreted to indicate that the opposing action of both motors mediates the normal distribution of neurofilaments along axons. Herein, we demonstrate that, while expression of relatively low levels of exogenous dynamitin indeed accelerated anterograde neurofilament transport along axonal neurites in culture, expression of progressively increasing levels of dynamitin induced focal accumulation of neurofilaments within axonal neurites and eventually caused neurite retraction. Inhibition of kinesin inhibited anterograde transport, but did not induce similar focal accumulations. These findings are consistent with studies indicating that perturbations in dynein activity can contribute to the aberrant accumulations of neurofilaments that accompany ALS/motor neuron disease.

  6. Impact of repeated stress on traumatic brain injury-induced mitochondrial electron transport chain expression and behavioral responses in rats.

    PubMed

    Xing, Guoqiang; Barry, Erin S; Benford, Brandi; Grunberg, Neil E; Li, He; Watson, William D; Sharma, Pushpa

    2013-01-01

    A significant proportion of the military personnel returning from Iraq and Afghanistan conflicts have suffered from both mild traumatic brain injury (mTBI) and post-traumatic stress disorder. The mechanisms are unknown. We used a rat model of repeated stress and mTBI to examine brain activity and behavioral function. Adult male Sprague-Dawley rats were divided into four groups: Naïve; 3 days repeated tail-shock stress; lateral fluid percussion mTBI; and repeated stress followed by mTBI (S-mTBI). Open field activity, sensorimotor responses, and acoustic startle responses (ASRs) were measured at various time points after mTBI. The protein expression of mitochondrial electron transport chain (ETC) complex subunits (CI-V) and pyruvate dehydrogenase (PDHE1α1) were determined in four brain regions at day 7-post mTBI. Compared to Naïves, repeated stress decreased horizontal activity; repeated stress and mTBI both decreased vertical activity; and the mTBI and S-mTBI groups were impaired in sensorimotor and ASRs. Repeated stress significantly increased CI, CII, and CIII protein levels in the prefrontal cortex (PFC), but decreased PDHE1α1 protein in the PFC and cerebellum, and decreased CIV protein in the hippocampus. The mTBI treatment decreased CV protein levels in the ipsilateral hippocampus. The S-mTBI treatment resulted in increased CII, CIII, CIV, and CV protein levels in the PFC, increased CI level in the cerebellum, and increased CIII and CV levels in the cerebral cortex, but decreased CI, CII, CIV, and PDHE1α1 protein levels in the hippocampus. Thus, repeated stress or mTBI alone differentially altered ETC expression in heterogeneous brain regions. Repeated stress followed by mTBI had synergistic effects on brain ETC expression, and resulted in more severe behavioral deficits. These results suggest that repeated stress could have contributed to the high incidence of long-term neurologic and neuropsychiatric morbidity in military personnel with or without mTBI.

  7. Axonal Localization of Integrins in the CNS Is Neuronal Type and Age Dependent.

    PubMed

    Andrews, Melissa R; Soleman, Sara; Cheah, Menghon; Tumbarello, David A; Mason, Matthew R J; Moloney, Elizabeth; Verhaagen, Joost; Bensadoun, Jean-Charles; Schneider, Bernard; Aebischer, Patrick; Fawcett, James W

    2016-01-01

    The regenerative ability of CNS axons decreases with age, however, this ability remains largely intact in PNS axons throughout adulthood. These differences are likely to correspond with age-related silencing of proteins necessary for axon growth and elongation. In previous studies, it has been shown that reintroduction of the α9 integrin subunit (tenascin-C receptor, α9) that is downregulated in adult CNS can improve neurite outgrowth and sensory axon regeneration after a dorsal rhizotomy or a dorsal column crush spinal cord lesion. In the current study, we demonstrate that virally expressed integrins (α9, α6, or β1 integrin) in the adult rat sensorimotor cortex and adult red nucleus are excluded from axons following neuronal transduction. Attempts to stimulate transport by inclusion of a cervical spinal injury and thus an upregulation of extracellular matrix molecules at the lesion site, or cotransduction with its binding partner, β1 integrin, did not induce integrin localization within axons. In contrast, virally expressed α9 integrin in developing rat cortex (postnatal day 5 or 10) demonstrated clear localization of integrins in cortical axons revealed by the presence of integrin in the axons of the corpus callosum and internal capsule, as well as in the neuronal cell body. Furthermore, examination of dorsal root ganglia neurons and retinal ganglion cells demonstrated integrin localization both within peripheral nerve as well as dorsal root axons and within optic nerve axons, respectively. Together, our results suggest a differential ability for in vivo axonal transport of transmembrane proteins dependent on neuronal age and subtype. PMID:27570822

  8. The axon as a physical structure in health and acute trauma.

    PubMed

    Kirkcaldie, Matthew T K; Collins, Jessica M

    2016-10-01

    The physical structure of neurons - dendrites converging on the soma, with an axon conveying activity to distant locations - is uniquely tied to their function. To perform their role, axons need to maintain structural precision in the soft, gelatinous environment of the central nervous system and the dynamic, flexible paths of nerves in the periphery. This requires close mechanical coupling between axons and the surrounding tissue, as well as an elastic, robust axoplasm resistant to pinching and flattening, and capable of sustaining transport despite physical distortion. These mechanical properties arise primarily from the properties of the internal cytoskeleton, coupled to the axonal membrane and the extracellular matrix. In particular, the two large constituents of the internal cytoskeleton, microtubules and neurofilaments, are braced against each other and flexibly interlinked by specialised proteins. Recent evidence suggests that the primary function of neurofilament sidearms is to structure the axoplasm into a linearly organised, elastic gel. This provides support and structure to the contents of axons in peripheral nerves subject to bending, protecting the relatively brittle microtubule bundles and maintaining them as transport conduits. Furthermore, a substantial proportion of axons are myelinated, and this thick jacket of membrane wrappings alters the form, function and internal composition of the axons to which it is applied. Together these structures determine the physical properties and integrity of neural tissue, both under conditions of normal movement, and in response to physical trauma. The effects of traumatic injury are directly dependent on the physical properties of neural tissue, especially axons, and because of axons' extreme structural specialisation, post-traumatic effects are usually characterised by particular modes of axonal damage. The physical realities of axons in neural tissue are integral to both normal function and their response to

  9. Axonal Localization of Integrins in the CNS Is Neuronal Type and Age Dependent

    PubMed Central

    Soleman, Sara; Mason, Matthew R. J.; Verhaagen, Joost; Bensadoun, Jean-Charles; Aebischer, Patrick

    2016-01-01

    The regenerative ability of CNS axons decreases with age, however, this ability remains largely intact in PNS axons throughout adulthood. These differences are likely to correspond with age-related silencing of proteins necessary for axon growth and elongation. In previous studies, it has been shown that reintroduction of the α9 integrin subunit (tenascin-C receptor, α9) that is downregulated in adult CNS can improve neurite outgrowth and sensory axon regeneration after a dorsal rhizotomy or a dorsal column crush spinal cord lesion. In the current study, we demonstrate that virally expressed integrins (α9, α6, or β1 integrin) in the adult rat sensorimotor cortex and adult red nucleus are excluded from axons following neuronal transduction. Attempts to stimulate transport by inclusion of a cervical spinal injury and thus an upregulation of extracellular matrix molecules at the lesion site, or cotransduction with its binding partner, β1 integrin, did not induce integrin localization within axons. In contrast, virally expressed α9 integrin in developing rat cortex (postnatal day 5 or 10) demonstrated clear localization of integrins in cortical axons revealed by the presence of integrin in the axons of the corpus callosum and internal capsule, as well as in the neuronal cell body. Furthermore, examination of dorsal root ganglia neurons and retinal ganglion cells demonstrated integrin localization both within peripheral nerve as well as dorsal root axons and within optic nerve axons, respectively. Together, our results suggest a differential ability for in vivo axonal transport of transmembrane proteins dependent on neuronal age and subtype. PMID:27570822

  10. The T1 domain of Kv1.3 mediates intracellular targeting to axons.

    PubMed

    Rivera, Jacqueline F; Chu, Po-Ju; Arnold, Don B

    2005-10-01

    Shaker K+ channels play an important role in modulating electrical excitability of axons. Recent work has demonstrated that the T1 tetramerization domain of Kv1.2 is both necessary and sufficient for targeting of the channel to the axonal surface [Gu, C., Jan, Y.N. & Jan, L.Y. (2003) Science,301, 646-649]. Here we use a related channel, Kv1.3, as a model to investigate cellular mechanisms that mediate axonal targeting. We show that the T1 domain of Kv1.3 is necessary and sufficient to mediate targeting of the channel to the axonal surface in pyramidal neurons in slices of cortex from neonatal rat. The T1 domain is also sufficient to cause preferential axonal localization of intracellular protein, which indicates that the domain probably does not work through compartment-specific endocytosis or compartment-specific vesicle docking. To determine whether the T1 domain mediates axonal trafficking of transport vesicles, we compared the trafficking of vesicles containing green fluorescent protein-labelled transferrin receptor with those containing the same protein fused with the T1 domain in living cortical neurons. Vesicles containing the wild-type transferrin receptor did not traffic to the axon, in accord with previously published results; however, those containing the transferrin receptor fused to T1 did traffic to the axon. These results are consistent with the T1 domain of Kv1.3 mediating axonal targeting by causing transport vesicles to traffic to axons and they represent the first evidence that such a mechanism might underlie axonal targeting. PMID:16262625

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

    SciTech Connect

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

    2011-01-05

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

  12. Initiation of Electron Transport Chain Activity in the Embryonic Heart Coincides with the Activation of Mitochondrial Complex 1 and the Formation of Supercomplexes

    PubMed Central

    Beutner, Gisela; Eliseev, Roman A.; Porter, George A.

    2014-01-01

    Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes. PMID:25427064

  13. Bicyclic-Capped Histone Deacetylase 6 Inhibitors with Improved Activity in a Model of Axonal Charcot-Marie-Tooth Disease.

    PubMed

    Shen, Sida; Benoy, Veronick; Bergman, Joel A; Kalin, Jay H; Frojuello, Mariana; Vistoli, Giulio; Haeck, Wanda; Van Den Bosch, Ludo; Kozikowski, Alan P

    2016-02-17

    Charcot-Marie-Tooth (CMT) disease is a disorder of the peripheral nervous system where progressive degeneration of motor and sensory nerves leads to motor problems and sensory loss and for which no pharmacological treatment is available. Recently, it has been shown in a model for the axonal form of CMT that histone deacetylase 6 (HDAC6) can serve as a target for the development of a pharmacological therapy. Therefore, we aimed at developing new selective and activity-specific HDAC6 inhibitors with improved biochemical properties. By utilizing a bicyclic cap as the structural scaffold from which to build upon, we developed several analogues that showed improved potency compared to tubastatin A while maintaining excellent selectivity compared to HDAC1. Further screening in N2a cells examining both the acetylation of α-tubulin and histones narrowed down the library of compounds to three potent and selective HDAC6 inhibitors. In mutant HSPB1-expressing DRG neurons, serving as an in vitro model for CMT2, these inhibitors were able to restore the mitochondrial axonal transport deficits. Combining structure-based development of HDAC6 inhibitors, screening in N2a cells and in a neuronal model for CMT2F, and preliminary ADMET and pharmacokinetic profiles, resulted in the selection of compound 23d that possesses improved biochemical, functional, and druglike properties compared to tubastatin A.

  14. Mechanisms of Distal Axonal Degeneration in Peripheral Neuropathies

    PubMed Central

    Cashman, Christopher R.; Höke, Ahmet

    2015-01-01

    Peripheral neuropathy is a common complication of a variety of diseases and treatments, including diabetes, cancer chemotherapy, and infectious causes (HIV, hepatitis C, and Campylobacter jejuni). Despite the fundamental difference between these insults, peripheral neuropathy develops as a combination of just six primary mechanisms: altered metabolism, covalent modification, altered organelle function and reactive oxygen species formation, altered intracellular and inflammatory signaling, slowed axonal transport, and altered ion channel dynamics and expression. All of these pathways converge to lead to axon dysfunction and symptoms of neuropathy. The detailed mechanisms of axon degeneration itself have begun to be elucidated with studies of animal models with altered degeneration kinetics, including the slowed Wallerian degeneration (Wlds) and Sarmknockout animal models. These studies have shown axonal degeneration to occur througha programmed pathway of injury signaling and cytoskeletal degradation. Insights into the common disease insults that converge on the axonal degeneration pathway promise to facilitate the development of therapeutics that may be effective against other mechanisms of neurodegeneration. PMID:25617478

  15. Mechanisms of distal axonal degeneration in peripheral neuropathies.

    PubMed

    Cashman, Christopher R; Höke, Ahmet

    2015-06-01

    Peripheral neuropathy is a common complication of a variety of diseases and treatments, including diabetes, cancer chemotherapy, and infectious causes (HIV, hepatitis C, and Campylobacter jejuni). Despite the fundamental difference between these insults, peripheral neuropathy develops as a combination of just six primary mechanisms: altered metabolism, covalent modification, altered organelle function and reactive oxygen species formation, altered intracellular and inflammatory signaling, slowed axonal transport, and altered ion channel dynamics and expression. All of these pathways converge to lead to axon dysfunction and symptoms of neuropathy. The detailed mechanisms of axon degeneration itself have begun to be elucidated with studies of animal models with altered degeneration kinetics, including the slowed Wallerian degeneration (Wld(S)) and Sarm knockout animal models. These studies have shown axonal degeneration to occur through a programmed pathway of injury signaling and cytoskeletal degradation. Insights into the common disease insults that converge on the axonal degeneration pathway promise to facilitate the development of therapeutics that may be effective against other mechanisms of neurodegeneration.

  16. Mechanisms of distal axonal degeneration in peripheral neuropathies.

    PubMed

    Cashman, Christopher R; Höke, Ahmet

    2015-06-01

    Peripheral neuropathy is a common complication of a variety of diseases and treatments, including diabetes, cancer chemotherapy, and infectious causes (HIV, hepatitis C, and Campylobacter jejuni). Despite the fundamental difference between these insults, peripheral neuropathy develops as a combination of just six primary mechanisms: altered metabolism, covalent modification, altered organelle function and reactive oxygen species formation, altered intracellular and inflammatory signaling, slowed axonal transport, and altered ion channel dynamics and expression. All of these pathways converge to lead to axon dysfunction and symptoms of neuropathy. The detailed mechanisms of axon degeneration itself have begun to be elucidated with studies of animal models with altered degeneration kinetics, including the slowed Wallerian degeneration (Wld(S)) and Sarm knockout animal models. These studies have shown axonal degeneration to occur through a programmed pathway of injury signaling and cytoskeletal degradation. Insights into the common disease insults that converge on the axonal degeneration pathway promise to facilitate the development of therapeutics that may be effective against other mechanisms of neurodegeneration. PMID:25617478

  17. ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis

    PubMed Central

    Konopacki, Filip A.; Dwivedy, Asha; Bellon, Anaïs; Blower, Michael D.

    2016-01-01

    Endocytosis and local protein synthesis (LPS) act coordinately to mediate the chemotropic responses of axons, but the link between these two processes is poorly understood. The endosomal sorting complex required for transport (ESCRT) is a key regulator of cargo sorting in the endocytic pathway, and here we have investigated the role of ESCRT-II, a critical ESCRT component, in Xenopus retinal ganglion cell (RGC) axons. We show that ESCRT-II is present in RGC axonal growth cones (GCs) where it co-localizes with endocytic vesicle GTPases and, unexpectedly, with the Netrin-1 receptor, deleted in colorectal cancer (DCC). ESCRT-II knockdown (KD) decreases endocytosis and, strikingly, reduces DCC in GCs and leads to axon growth and guidance defects. ESCRT-II-depleted axons fail to turn in response to a Netrin-1 gradient in vitro and many axons fail to exit the eye in vivo. These defects, similar to Netrin-1/DCC loss-of-function phenotypes, can be rescued in whole (in vitro) or in part (in vivo) by expressing DCC. In addition, ESCRT-II KD impairs LPS in GCs and live imaging reveals that ESCRT-II transports mRNAs in axons. Collectively, our results show that the ESCRT-II-mediated endocytic pathway regulates both DCC and LPS in the axonal compartment and suggest that ESCRT-II aids gradient sensing in GCs by coupling endocytosis to LPS. PMID:27248654

  18. Alterations in mitochondrial electron transport system activity in response to warm acclimation, hypoxia-reoxygenation and copper in rainbow trout, Oncorhynchus mykiss.

    PubMed

    Sappal, Ravinder; MacDougald, Michelle; Fast, Mark; Stevens, Don; Kibenge, Fred; Siah, Ahmed; Kamunde, Collins

    2015-08-01

    Fish expend significant amounts of energy to handle the numerous potentially stressful biotic and abiotic factors that they commonly encounter in aquatic environments. This universal requirement for energy singularizes mitochondria, the primary cellular energy transformers, as fundamental drivers of responses to environmental change. Our study probed the interacting effects of thermal stress, hypoxia-reoxygenation (HRO) and copper (Cu) exposure in rainbow trout to test the prediction that they act jointly to impair mitochondrial function. Rainbow trout were acclimated to 11 (controls) or 20°C for 2 months. Liver mitochondria were then isolated and their responses in vitro to Cu (0-20μM) without and with HRO were assessed. Sequential inhibition and activation of mitochondrial electron transport system (ETS) enzyme complexes permitted the measurement of respiratory activities supported by complex I-IV (CI-IV) in one run. The results showed that warm acclimation reduced fish and liver weights but increased mitochondrial protein indicating impairment of energy metabolism, increased synthesis of defense proteins and/or reduced liver water content. Whereas acute rise (11→20°C) in temperature increased mitochondrial oxidation rates supported by CI-IV, warm acclimation reduced the maximal (state 3) and increased the basal (state 4) respiration leading to global uncoupling of oxidative phosphorylation (OXPHOS). HRO profoundly inhibited both maximal and basal respiration rates supported by CI-IV, reduced RCR for all except CII and lowered CI:CII respiration ratio, an indication of decreased OXPHOS efficiency. The effects of Cu were less pronounced but more variable and included inhibition of CII-IV maximal respiration rates and stimulation of both CI and CIII basal respiration rates. Surprisingly, only CII and CIII indices exhibited significant 3-way interactions whereas 2-way interactions of acclimation either with Cu or HRO were portrayed mostly by CIV, and those of

  19. Mitochondrial fusion but not fission regulates larval growth and synaptic development through steroid hormone production

    PubMed Central

    Sandoval, Hector; Yao, Chi-Kuang; Chen, Kuchuan; Jaiswal, Manish; Donti, Taraka; Lin, Yong Qi; Bayat, Vafa; Xiong, Bo; Zhang, Ke; David, Gabriela; Charng, Wu-Lin; Yamamoto, Shinya; Duraine, Lita; Graham, Brett H; Bellen, Hugo J

    2014-01-01

    Mitochondrial fusion and fission affect the distribution and quality control of mitochondria. We show that Marf (Mitochondrial associated regulatory factor), is required for mitochondrial fusion and transport in long axons. Moreover, loss of Marf leads to a severe depletion of mitochondria in neuromuscular junctions (NMJs). Marf mutants also fail to maintain proper synaptic transmission at NMJs upon repetitive stimulation, similar to Drp1 fission mutants. However, unlike Drp1, loss of Marf leads to NMJ morphology defects and extended larval lifespan. Marf is required to form contacts between the endoplasmic reticulum and/or lipid droplets (LDs) and for proper storage of cholesterol and ecdysone synthesis in ring glands. Interestingly, human Mitofusin-2 rescues the loss of LD but both Mitofusin-1 and Mitofusin-2 are required for steroid-hormone synthesis. Our data show that Marf and Mitofusins share an evolutionarily conserved role in mitochondrial transport, cholesterol ester storage and steroid-hormone synthesis. DOI: http://dx.doi.org/10.7554/eLife.03558.001 PMID:25313867

  20. Oligodendrocytes: Myelination and Axonal Support.

    PubMed

    Simons, Mikael; Nave, Klaus-Armin

    2015-06-22

    Myelinated nerve fibers have evolved to enable fast and efficient transduction of electrical signals in the nervous system. To act as an electric insulator, the myelin sheath is formed as a multilamellar membrane structure by the spiral wrapping and subsequent compaction of the oligodendroglial plasma membrane around central nervous system (CNS) axons. Current evidence indicates that the myelin sheath is more than an inert insulating membrane structure. Oligodendrocytes are metabolically active and functionally connected to the subjacent axon via cytoplasmic-rich myelinic channels for movement of macromolecules to and from the internodal periaxonal space under the myelin sheath. This review summarizes our current understanding of how myelin is generated and also the role of oligodendrocytes in supporting the long-term integrity of myelinated axons.

  1. Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate.

    PubMed

    Miyayama, Takamitsu; Arai, Yuta; Suzuki, Noriyuki; Hirano, Seishiro

    2013-03-01

    Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage.

  2. Comparison of Intact Arabidopsis thaliana Leaf Transcript Profiles during Treatment with Inhibitors of Mitochondrial Electron Transport and TCA Cycle

    PubMed Central

    Yu, Jianping; Ruckle, Michael E.; McIntosh, Lee; Hock, Jeffery J.; Bingham, Scott; White, Samuel J.; George, Rajani M.; Subbaiah, Chalivendra C.; Rhoads, David M.

    2012-01-01

    Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress. PMID:23028523

  3. Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs).

    PubMed

    Rueda, Carlos B; Llorente-Folch, Irene; Traba, Javier; Amigo, Ignacio; Gonzalez-Sanchez, Paloma; Contreras, Laura; Juaristi, Inés; Martinez-Valero, Paula; Pardo, Beatriz; Del Arco, Araceli; Satrustegui, Jorgina

    2016-08-01

    Glutamate elicits Ca(2+) signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca(2+) activates respiration by stimulation of the NADH malate-aspartate shuttle through Ca(2+)-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca(2+) binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca(2+) entry into the matrix through the mitochondrial Ca(2+) uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca(2+) overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca(2+) deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD(+), a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca(2+)-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca(2+) entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca(2+), resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar

  4. Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier

    PubMed Central

    King, Martin S.; Kerr, Matthew; Crichton, Paul G.; Springett, Roger; Kunji, Edmund R.S.

    2016-01-01

    Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway. PMID:26453935

  5. Aquaporin-1 water permeability as a novel determinant of axonal regeneration in dorsal root ganglion neurons.

    PubMed

    Zhang, Hua; Verkman, A S

    2015-03-01

    Dorsal root ganglion (DRG) neurons transduce peripheral pain signals through small-diameter, non-myelinated C-fibers, which, when injured, can regenerate to restore pain sensation. Water channel aquaporin-1 (AQP1) is expressed at the plasma membrane of cell bodies and axons of DRG neurons, where it modulates the sensing of certain types of pain. Here, we found that AQP1 is also involved in DRG axonal growth and regeneration by a mechanism that may involve water transport-facilitated extension of axonal outgrowths. Spontaneous and nerve growth factor-stimulated axonal extension was reduced in cultures of AQP1-deficient DRG neurons and DRG explants compared to the wildtype. Axonal growth in AQP1-deficient DRG cultures was rescued by transfection with AQP1 or a different water-transporting AQP (AQP4), but not by a non-water-transporting AQP1 mutant. Following sciatic nerve compression injury AQP1 expression was increased in DRG neurons in wildtype mice, and DRG axonal growth was impaired in AQP1-deficient mice. Our results indicate AQP1 as a novel determinant of DRG axonal regeneration and hence a potential therapeutic target to accelerate neuronal regeneration.

  6. AQUAPORIN-1 WATER PERMEABILITY AS A NOVEL DETERMINANT OF AXONAL REGENERATION IN DORSAL ROOT GANGLION NEURONS

    PubMed Central

    Zhang, Hua; Verkman, A.S.

    2015-01-01

    Dorsal root ganglion (DRG) neurons transduce peripheral pain signals through small-diameter, non-myelinated C-fibers, which, when injured, can regenerate to restore pain sensation. Water channel aquaporin-1 (AQP1) is expressed at the plasma membrane of cell bodies and axons of DRG neurons, where it modulates the sensing of certain types of pain. Here, we found that AQP1 is also involved in DRG axonal growth and regeneration by a mechanism that may involve water transport-facilitated extension of axonal outgrowths. Spontaneous and nerve growth factor-stimulated axonal extension was reduced in cultures of AQP1-deficient DRG neurons and DRG explants compared to the wildtype. Axonal growth in AQP1-deficient DRG cultures was rescued by transfection with AQP1 or a different water-transporting AQP (AQP4), but not by a non-water-transporting AQP1 mutant. Following sciatic nerve compression injury AQP1 expression was increased in DRG neurons in wildtype mice, and DRG axonal growth was impaired in AQP1-deficient mice. Our results indicate AQP1 as a novel determinant of DRG axonal regeneration and hence a potential therapeutic target to accelerate neuronal regeneration. PMID:25585012

  7. Axon-axon interactions in neuronal circuit assembly: lessons from olfactory map formation.

    PubMed

    Imai, Takeshi; Sakano, Hitoshi

    2011-11-01

    During the development of the nervous system, neurons often connect axons and dendrites over long distances, which are navigated by chemical cues. During the past few decades, studies on axon guidance have focused on chemical cues provided by the axonal target or intermediate target. However, recent studies have shed light on the roles and mechanisms underlying axon-axon interactions during neuronal circuit assembly. The roles of axon-axon interactions are best exemplified in recent studies on olfactory map formation in vertebrates. Pioneer-follower interaction is essential for the axonal pathfinding process. Pre-target axon sorting establishes the anterior-posterior map order. The temporal order of axonal projection is converted to dorsal-ventral topography with the aid of secreted molecules provided by early-arriving axons. An activity-dependent process to form a discrete map also depends on axon sorting. Thus, an emerging principle of olfactory map formation is the 'self-organisation' of axons rather than the 'lock and key' matching between axons and targets. In this review, we discuss how axon-axon interactions contribute to neuronal circuit assembly. PMID:22103421

  8. Synthesis of sodium channels in the cell bodies of squid giant axons.

    PubMed Central

    Brismar, T; Gilly, W F

    1987-01-01

    Giant axons in squid are formed by fusion of axons from many small cell bodies in the giant fiber lobe (GFL) of the stellate ganglion. Somata of GFL cells in vivo are inexcitable and do not have measurable sodium current (INa) when studied with microelectrode or patch-electrode voltage-clamp techniques. If GFL cells are separated from the giant axons and maintained in primary culture, axon-like INa can be recorded from the somata after several days. Incorporation of Na channels into GFL cell bodies requires protein synthesis, intracellular microtubule-based transport, and the lack of a morphologically defined axon to serve as a sink for channels synthesized in culture. PMID:3469679

  9. Pyrroloquinoline Quinone Resists Denervation-Induced Skeletal Muscle Atrophy by Activating PGC-1α and Integrating Mitochondrial Electron Transport Chain Complexes

    PubMed Central

    Kuo, Yung-Ting; Shih, Ping-Hsiao; Kao, Shu-Huei; Yeh, Geng-Chang; Lee, Horng-Mo

    2015-01-01

    Denervation-mediated skeletal muscle atrophy results from the loss of electric stimulation and leads to protein degradation, which is critically regulated by the well-confirmed transcriptional co-activator peroxisome proliferator co-activator 1 alpha (PGC-1α). No adequate treatments of muscle wasting are available. Pyrroloquinoline quinone (PQQ), a naturally occurring antioxidant component with multiple functions including mitochondrial modulation, demonstrates the ability to protect against muscle dysfunction. However, it remains unclear whether PQQ enhances PGC-1α activation and resists skeletal muscle atrophy in mice subjected to a denervation operation. This work investigates the expression of PGC-1α and mitochondrial function in the skeletal muscle of denervated mice administered PQQ. The C57BL6/J mouse was subjected to a hindlimb sciatic axotomy. A PQQ-containing ALZET® osmotic pump (equivalent to 4.5 mg/day/kg b.w.) was implanted subcutaneously into the right lower abdomen of the mouse. In the time course study, the mouse was sacrificed and the gastrocnemius muscle was prepared for further myopathological staining, energy metabolism analysis, western blotting, and real-time quantitative PCR studies. We observed that PQQ administration abolished the denervation-induced decrease in muscle mass and reduced mitochondrial activities, as evidenced by the reduced fiber size and the decreased expression of cytochrome c oxidase and NADH-tetrazolium reductase. Bioenergetic analysis demonstrated that PQQ reprogrammed the denervation-induced increase in the mitochondrial oxygen consumption rate (OCR) and led to an increase in the extracellular acidification rate (ECAR), a measurement of the glycolytic metabolism. The protein levels of PGC-1α and the electron transport chain (ETC) complexes were also increased by treatment with PQQ. Furthermore, PQQ administration highly enhanced the expression of oxidative fibers and maintained the type II glycolytic fibers. This

  10. Pyrroloquinoline Quinone Resists Denervation-Induced Skeletal Muscle Atrophy by Activating PGC-1α and Integrating Mitochondrial Electron Transport Chain Complexes.

    PubMed

    Kuo, Yung-Ting; Shih, Ping-Hsiao; Kao, Shu-Huei; Yeh, Geng-Chang; Lee, Horng-Mo

    2015-01-01

    Denervation-mediated skeletal muscle atrophy results from the loss of electric stimulation and leads to protein degradation, which is critically regulated by the well-confirmed transcriptional co-activator peroxisome proliferator co-activator 1 alpha (PGC-1α). No adequate treatments of muscle wasting are available. Pyrroloquinoline quinone (PQQ), a naturally occurring antioxidant component with multiple functions including mitochondrial modulation, demonstrates the ability to protect against muscle dysfunction. However, it remains unclear whether PQQ enhances PGC-1α activation and resists skeletal muscle atrophy in mice subjected to a denervation operation. This work investigates the expression of PGC-1α and mitochondrial function in the skeletal muscle of denervated mice administered PQQ. The C57BL6/J mouse was subjected to a hindlimb sciatic axotomy. A PQQ-containing ALZET® osmotic pump (equivalent to 4.5 mg/day/kg b.w.) was implanted subcutaneously into the right lower abdomen of the mouse. In the time course study, the mouse was sacrificed and the gastrocnemius muscle was prepared for further myopathological staining, energy metabolism analysis, western blotting, and real-time quantitative PCR studies. We observed that PQQ administration abolished the denervation-induced decrease in muscle mass and reduced mitochondrial activities, as evidenced by the reduced fiber size and the decreased expression of cytochrome c oxidase and NADH-tetrazolium reductase. Bioenergetic analysis demonstrated that PQQ reprogrammed the denervation-induced increase in the mitochondrial oxygen consumption rate (OCR) and led to an increase in the extracellular acidification rate (ECAR), a measurement of the glycolytic metabolism. The protein levels of PGC-1α and the electron transport chain (ETC) complexes were also increased by treatment with PQQ. Furthermore, PQQ administration highly enhanced the expression of oxidative fibers and maintained the type II glycolytic fibers. This

  11. Pyrroloquinoline Quinone Resists Denervation-Induced Skeletal Muscle Atrophy by Activating PGC-1α and Integrating Mitochondrial Electron Transport Chain Complexes.

    PubMed

    Kuo, Yung-Ting; Shih, Ping-Hsiao; Kao, Shu-Huei; Yeh, Geng-Chang; Lee, Horng-Mo

    2015-01-01

    Denervation-mediated skeletal muscle atrophy results from the loss of electric stimulation and leads to protein degradation, which is critically regulated by the well-confirmed transcriptional co-activator peroxisome proliferator co-activator 1 alpha (PGC-1α). No adequate treatments of muscle wasting are available. Pyrroloquinoline quinone (PQQ), a naturally occurring antioxidant component with multiple functions including mitochondrial modulation, demonstrates the ability to protect against muscle dysfunction. However, it remains unclear whether PQQ enhances PGC-1α activation and resists skeletal muscle atrophy in mice subjected to a denervation operation. This work investigates the expression of PGC-1α and mitochondrial function in the skeletal muscle of denervated mice administered PQQ. The C57BL6/J mouse was subjected to a hindlimb sciatic axotomy. A PQQ-containing ALZET® osmotic pump (equivalent to 4.5 mg/day/kg b.w.) was implanted subcutaneously into the right lower abdomen of the mouse. In the time course study, the mouse was sacrificed and the gastrocnemius muscle was prepared for further myopathological staining, energy metabolism analysis, western blotting, and real-time quantitative PCR studies. We observed that PQQ administration abolished the denervation-induced decrease in muscle mass and reduced mitochondrial activities, as evidenced by the reduced fiber size and the decreased expression of cytochrome c oxidase and NADH-tetrazolium reductase. Bioenergetic analysis demonstrated that PQQ reprogrammed the denervation-induced increase in the mitochondrial oxygen consumption rate (OCR) and led to an increase in the extracellular acidification rate (ECAR), a measurement of the glycolytic metabolism. The protein levels of PGC-1α and the electron transport chain (ETC) complexes were also increased by treatment with PQQ. Furthermore, PQQ administration highly enhanced the expression of oxidative fibers and maintained the type II glycolytic fibers. This

  12. Loss of Modifier of Cell Adhesion Reveals a Pathway Leading to Axonal Degeneration

    PubMed Central

    Chen, Qi; Peto, Charles A.; Shelton, G. Diane; Mizisin, Andrew; Sawchenko, Paul E.; Schubert, David

    2009-01-01

    Axonal dysfunction is the major phenotypic change in many neurodegenerative diseases, but the processes underlying this impairment are not clear. Modifier of cell adhesion (MOCA) is a presenilin binding protein that functions as a guanine nucleotide exchange factor for Rac1. The loss of MOCA in mice leads to axonal degeneration and causes sensorimotor impairments by decreasing cofilin phosphorylation and altering its upstream signaling partners LIM kinase and p21-activated kinase, an enzyme directly downstream of Rac1. The dystrophic axons found in MOCA-deficient mice are associated with abnormal aggregates of neurofilament protein, the disorganization of the axonal cytoskeleton, and the accumulation of autophagic vacuoles and polyubiquitinated proteins. Furthermore, MOCA deficiency causes an alteration in the actin cytoskeleton and the formation of cofilin-containing rod-like structures. The dystrophic axons show functional abnormalities, including impaired axonal transport. These findings demonstrate that MOCA is required for maintaining the functional integrity of axons and define a model for the steps leading to axonal degeneration. PMID:19129390

  13. The mixture of "ecstasy" and its metabolites impairs mitochondrial fusion/fission equilibrium and trafficking in hippocampal neurons, at in vivo relevant concentrations.

    PubMed

    Barbosa, Daniel José; Serrat, Romàn; Mirra, Serena; Quevedo, Martí; de Barreda, Elena Goméz; Àvila, Jesús; Ferreira, Luísa Maria; Branco, Paula Sério; Fernandes, Eduarda; Lourdes Bastos, Maria de; Capela, João Paulo; Soriano, Eduardo; Carvalho, Félix

    2014-06-01

    3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a potentially neurotoxic recreational drug of abuse. Though the mechanisms involved are still not completely understood, formation of reactive metabolites and mitochondrial dysfunction contribute to MDMA-related neurotoxicity. Neuronal mitochondrial trafficking, and their targeting to synapses, is essential for proper neuronal function and survival, rendering neurons particularly vulnerable to mitochondrial dysfunction. Indeed, MDMA-associated disruption of Ca(2+) homeostasis and ATP depletion have been described in neurons, thus suggesting possible MDMA interference on mitochondrial dynamics. In this study, we performed real-time functional experiments of mitochondrial trafficking to explore the role of in situ mitochondrial dysfunction in MDMA's neurotoxic actions. We show that the mixture of MDMA and six of its major in vivo metabolites, each compound at 10μM, impaired mitochondrial trafficking and increased the fragmentation of axonal mitochondria in cultured hippocampal neurons. Furthermore, the overexpression of mitofusin 2 (Mfn2) or dynamin-related protein 1 (Drp1) K38A constructs almost completely rescued the trafficking deficits caused by this mixture. Finally, in hippocampal neurons overexpressing a Mfn2 mutant, Mfn2 R94Q, with impaired fusion and transport properties, it was confirmed that a dysregulation of mitochondrial fission/fusion events greatly contributed to the reported trafficking phenotype. In conclusion, our study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at concentrations relevant to the in vivo scenario, impaired mitochondrial trafficking and increased mitochondrial fragmentation in hippocampal neurons, thus providing a new insight in the context of "ecstasy"-induced neuronal injury.

  14. Computer modeling of mild axonal injury: implications for axonal signal transmission.

    PubMed

    Volman, Vladislav; Ng, Laurel J

    2013-10-01

    Diffusion imaging and postmortem studies of patients with mild traumatic brain injury (mTBI) of the concussive type are consistent with the observations of diffuse axonal injury to the white matter axons. Mechanical trauma to axons affects the properties of tetrodotoxin-sensitive sodium channels at the nodes of Ranvier, leading to axonal degeneration through intra-axonal accumulation of calcium ions and activation of calcium proteases; however, the immediate implications of axonal trauma regarding axonal functionality and their relevance to transient impairment of function as observed in concussion remain elusive. A biophysically realistic computational model of a myelinated axon was developed to investigate how mTBI could immediately affect axonal function. Traumatized axons showed alterations in signal propagation properties that nonlinearly depended on the level of trauma; subthreshold traumatized axons had decreased spike propagation time, whereas suprathreshold traumatized axons exhibited a slowdown of spike propagation and spike propagation failure. Trauma had consistently reduced axonal spike amplitude. The susceptibility of an axon to trauma could be modulated by the function of an ATP-dependent sodium-potassium pump. The results suggest a mechanism by which concussive mTBI could lead to the immediate impairment of signal propagation through the axon and the emerging dysfunctional neuronal information exchange.

  15. A Putative Mitochondrial Iron Transporter MrsA in Aspergillus fumigatus Plays Important Roles in Azole-, Oxidative Stress Responses and Virulence.

    PubMed

    Long, Nanbiao; Xu, Xiaoling; Qian, Hui; Zhang, Shizhu; Lu, Ling

    2016-01-01

    Iron is an essential nutrient and enzyme co-factor required for a wide range of cellular processes, especially for the function of mitochondria. For the opportunistic fungal pathogen Aspergillus fumigatus, the ability to obtain iron is required for growth and virulence during the infection process. However, knowledge of how mitochondria are involved in iron regulation is still limited. Here, we show that a mitochondrial iron transporter, MrsA, a homolog of yeast Mrs4p, is critical for adaptation to iron-limited or iron-excess conditions in A. fumigatus. Deletion of mrsA leads to disruption of iron homeostasis with a decreased sreA expression, resulted in activated reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). Furthermore, deletion of mrsA induces hypersusceptibility to azole and oxidative stresses. An assay for cellular ROS content in ΔmrsA combined with rescue from the mrsA-defective phenotype by the antioxidant reagent L-ascorbic acid indicates that the increased sensitivity of ΔmrsA to the azole itraconazole and to oxidative stress is mainly the result of abnormal ROS accumulation. Moreover, site-directed mutation experiments verified that three conserved histidine residues related to iron transport in MrsA are required for responses to oxidative and azole stresses. Importantly, ΔmrsA causes significant attenuation of virulence in an immunocompromised murine model of aspergillosis. Collectively, our results show that the putative mitochondrial iron transporter MrsA plays important roles in azole- and oxidative-stress responses and virulence by regulating the balance of cellular iron in A. fumigatus. PMID:27433157

  16. A Putative Mitochondrial Iron Transporter MrsA in Aspergillus fumigatus Plays Important Roles in Azole-, Oxidative Stress Responses and Virulence

    PubMed Central

    Long, Nanbiao; Xu, Xiaoling; Qian, Hui; Zhang, Shizhu; Lu, Ling

    2016-01-01

    Iron is an essential nutrient and enzyme co-factor required for a wide range of cellular processes, especially for the function of mitochondria. For the opportunistic fungal pathogen Aspergillus fumigatus, the ability to obtain iron is required for growth and virulence during the infection process. However, knowledge of how mitochondria are involved in iron regulation is still limited. Here, we show that a mitochondrial iron transporter, MrsA, a homolog of yeast Mrs4p, is critical for adaptation to iron-limited or iron-excess conditions in A. fumigatus. Deletion of mrsA leads to disruption of iron homeostasis with a decreased sreA expression, resulted in activated reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). Furthermore, deletion of mrsA induces hypersusceptibility to azole and oxidative stresses. An assay for cellular ROS content in ΔmrsA combined with rescue from the mrsA-defective phenotype by the antioxidant reagent L-ascorbic acid indicates that the increased sensitivity of ΔmrsA to the azole itraconazole and to oxidative stress is mainly the result of abnormal ROS accumulation. Moreover, site-directed mutation experiments verified that three conserved histidine residues related to iron transport in MrsA are required for responses to oxidative and azole stresses. Importantly, ΔmrsA causes significant attenuation of virulence in an immunocompromised murine model of aspergillosis. Collectively, our results show that the putative mitochondrial iron transporter MrsA plays important roles in azole- and oxidative-stress responses and virulence by regulating the balance of cellular iron in A. fumigatus. PMID:27433157

  17. Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.

    PubMed

    Wallström, Sabá V; Florez-Sarasa, Igor; Araújo, Wagner L; Escobar, Matthew A; Geisler, Daniela A; Aidemark, Mari; Lager, Ida; Fernie, Alisdair R; Ribas-Carbó, Miquel; Rasmusson, Allan G

    2014-05-01

    The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)⁺ ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.

  18. Axon degeneration: context defines distinct pathways.

    PubMed

    Geden, Matthew J; Deshmukh, Mohanish

    2016-08-01

    Axon degeneration is an essential part of development, plasticity, and injury response and has been primarily studied in mammalian models in three contexts: 1) Axotomy-induced Wallerian degeneration, 2) Apoptosis-induced axon degeneration (axon apoptosis), and 3) Axon pruning. These three contexts dictate engagement of distinct pathways for axon degeneration. Recent advances have identified the importance of SARM1, NMNATs, NAD+ depletion, and MAPK signaling in axotomy-induced Wallerian degeneration. Interestingly, apoptosis-induced axon degeneration and axon pruning have many shared mechanisms both in signaling (e.g. DLK, JNKs, GSK3α/β) and execution (e.g. Puma, Bax, caspase-9, caspase-3). However, the specific mechanisms by which caspases are activated during apoptosis versus pruning appear distinct, with apoptosis requiring Apaf-1 but not caspase-6 while pruning requires caspase-6 but not Apaf-1. PMID:27197022

  19. Effect of coenzyme Q10 intake on endogenous coenzyme Q content, mitochondrial electron transport chain, antioxidative defenses, and life span of mice.

    PubMed

    Sohal, Rajindar S; Kamzalov, Sergey; Sumien, Nathalie; Ferguson, Melissa; Rebrin, Igor; Heinrich, Kevin R; Forster, Michael J

    2006-02-01

    The main purpose of this study was to determine whether intake of coenzyme Q10, which can potentially act as both an antioxidant and a prooxidant, has an impact on indicators of oxidative stress and the aging process. Mice were fed diets providing daily supplements of 0, 93, or 371 mg CoQ10 /kg body weight, starting at 3.5 months of age. Effects on mitochondrial superoxide generation, activities of oxidoreductases, protein oxidative damage, glutathione redox state, and life span of male mice were determined. Amounts of CoQ9 and CoQ10, measured after 3.5 or 17.5 months of intake, in homogenates and mitochondria of liver, heart, kidney, skeletal muscle, and brain increased with the dosage and duration of CoQ10 intake in all the tissues except brain. Activities of mitochondrial electron transport chain oxidoreductases, rates of mitochondrial O2-* generation, state 3 respiration, carbonyl content, glutathione redox state of tissues, and activities of superoxide dismutase, catalase, and glutathione peroxidase, determined at 19 or 25 months of age, were unaffected by CoQ10 administration. Life span studies, conducted on 50 mice in each group, showed that CoQ10 administration had no effect on mortality. Altogether, the results indicated that contrary to the historical view, supplemental intake of CoQ10 elevates the endogenous content of both CoQ9 and CoQ10, but has no discernable effect on the main antioxidant defenses or prooxidant generation in most tissues, and has no impact on the life span of mice.

  20. The development of mitochondrial medicine.

    PubMed Central

    Luft, R

    1994-01-01

    Primary defects in mitochondrial function are implicated in over 100 diseases, and the list continues to grow. Yet the first mitochondrial defect--a myopathy--was demonstrated only 35 years ago. The field's dramatic expansion reflects growth of knowledge in three areas: (i) characterization of mitochondrial structure and function, (ii) elucidation of the steps involved in mitochondrial biosynthesis, and (iii) discovery of specific mitochondrial DNA. Many mitochondrial diseases are accompanied by mutations in this DNA. Inheritance is by maternal transmission. The metabolic defects encompass the electron transport complexes, intermediates of the tricarboxylic acid cycle, and substrate transport. The clinical manifestations are protean, most often involving skeletal muscle and the central nervous system. In addition to being a primary cause of disease, mitochondrial DNA mutations and impaired oxidation have now been found to occur as secondary phenomena in aging as well as in age-related degenerative diseases such as Parkinson, Alzheimer, and Huntington diseases, amyotrophic lateral sclerosis and cardiomyopathies, atherosclerosis, and diabetes mellitus. Manifestations of both the primary and secondary mitochondrial diseases are thought to result from the production of oxygen free radicals. With increased understanding of the mechanisms underlying the mitochondrial dysfunctions has come the beginnings of therapeutic strategies, based mostly on the administration of antioxidants, replacement of cofactors, and provision of nutrients. At the present accelerating pace of development of what may be called mitochondrial medicine, much more is likely to be achieved within the next few years. Images PMID:8090715

  1. Calcium Efflux from Internally Dialyzed Squid Giant Axons

    PubMed Central

    Dipolo, Reinaldo

    1973-01-01

    Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 µM calcium and 5 mM ATP was 0.26 pmol/cm2·s at 22°C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca]i lower than 0.3µM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na]o and [Ca]o by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5µg/ml oligomycin, analysis of the perfusion effluent gave values of 1–4 µM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca]i, (80 µM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10-7 mol Ca++/mg of protein with an initial rate of 2.6 x 10-8 mol Ca++/min·mg of protein. Axons dialyzed with 2 mM cyanide after 8–10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources. PMID:4751386

  2. Dendrite and Axon Specific Geometrical Transformation in Neurite Development

    PubMed Central

    Mironov, Vasily I.; Semyanov, Alexey V.; Kazantsev, Victor B.

    2016-01-01

    We propose a model of neurite growth to explain the differences in dendrite and axon specific neurite development. The model implements basic molecular kinetics, e.g., building protein synthesis and transport to the growth cone, and includes explicit dependence of the building kinetics on the geometry of the neurite. The basic assumption was that the radius of the neurite decreases with length. We found that the neurite dynamics crucially depended on the relationship between the rate of active transport and the rate of morphological changes. If these rates were in the balance, then the neurite displayed axon specific development with a constant elongation speed. For dendrite specific growth, the maximal length was rapidly saturated by degradation of building protein structures or limited by proximal part expansion reaching the characteristic cell size. PMID:26858635

  3. Mitochondrial Diseases

    MedlinePlus

    ... disorder, something goes wrong with this process. Mitochondrial diseases are a group of metabolic disorders. Mitochondria are ... cells and cause damage. The symptoms of mitochondrial disease can vary. It depends on how many mitochondria ...

  4. Diabetic polyneuropathy. Axonal or demyelinating?

    PubMed

    Valls-Canals, J; Povedano, M; Montero, J; Pradas, J

    2002-01-01

    Diabetic polyneuropathy is the most common subgroup of diabetic neuropathy, but its nature is controversial as it might be demyelinating and/or axonal. We have tried to determine whether diabetic polyneuropathy is electrophysiologically axonal, demyelinating, or both. We have studied the sural and peroneal nerves and the electromyographies of leg muscles in 50 healthy subjects (average age 67.2 years, range 45 to 84 years), in 50 diabetic patients (average age 66.34 years, range 44 to 82 years) showing no symptoms and/or signs of polyneuropathy (DP1), and in 50 diabetic patients (average age 67.10 years, range 49 to 87 years) showing symptoms and/or signs of polyneuropathy (DP2). The amplitude (AMP) of sural and peroneal nerves in healthy and DP1 subjects was similar. Conduction velocity (CV) of sural and peroneal nerves was slower in DP1 subjects than in healthy subjects. DP2 subjects showed AMP and CV values significantly lower than those in DP1 subjects, and signs of acute and chronic denervation/reinervation were found in the leg muscles. We believe that this result indicates that diabetic patients have two types of polyneuropathies: a demyelinating disease that could appear in diabetic patients with and without symptoms of polyneuropathy, and an axonal loss that is responsible for most of the symptoms.

  5. mRNAs and Protein Synthetic Machinery Localize into Regenerating Spinal Cord Axons When They Are Provided a Substrate That Supports Growth

    PubMed Central

    Kalinski, Ashley L.; Sachdeva, Rahul; Gomes, Cynthia; Lee, Seung Joon; Shah, Zalak; Houle, John D.

    2015-01-01

    Although intra-axonal protein synthesis is well recognized in cultured neurons and during development in vivo, there have been few reports of mRNA localization and/or intra-axonal translation in mature CNS axons. Indeed, previous work indicated that mature CNS axons contain much lower quantities of translational machinery than PNS axons, leading to the conclusion that the capacity for intra-axonal protein synthesis is linked to the intrinsic capacity of a neuron for regeneration, with mature CNS neurons showing much less growth after injury than PNS neurons. However, when regeneration by CNS axons is facilitated, it is not known whether the intra-axonal content of translational machinery changes or whether mRNAs localize into these axons. Here, we have used a peripheral nerve segment grafted into the transected spinal cord of adult rats as a supportive environment for regeneration by ascending spinal axons. By quantitative fluorescent in situ hybridization combined with immunofluorescence to unambiguously distinguish intra-axonal mRNAs, we show that regenerating spinal cord axons contain β-actin, GAP-43, Neuritin, Reg3a, Hamp, and Importin β1 mRNAs. These axons also contain 5S rRNA, phosphorylated S6 ribosomal protein, eIF2α translation factor, and 4EBP1 translation factor inhibitory protein. Different levels of these mRNAs in CNS axons from regenerating PNS axons may relate to differences in the growth capacity of these neurons, although the presence of mRNA transport and likely local translation in both CNS and PNS neurons suggests an active role in the regenerative process. SIGNIFICANCE STATEMENT Although peripheral nerve axons retain the capacity to locally synthesize proteins into adulthood, previous studies have argued that mature brain and spinal cord axons cannot synthesize proteins. Protein synthesis in peripheral nerve axons is increased during regeneration, and intra-axonally synthesized proteins have been shown to contribute to nerve regeneration

  6. A Stochastic Multiscale Model That Explains the Segregation of Axonal Microtubules and Neurofilaments in Neurological Diseases

    PubMed Central

    Xue, Chuan; Shtylla, Blerta; Brown, Anthony

    2015-01-01

    The organization of the axonal cytoskeleton is a key determinant of the normal function of an axon, which is a long thin projection of a neuron. Under normal conditions two axonal cytoskeletal polymers, microtubules and neurofilaments, align longitudinally in axons and are interspersed in axonal cross-sections. However, in many neurotoxic and neurodegenerative disorders, microtubules and neurofilaments segregate apart from each other, with microtubules and membranous organelles clustered centrally and neurofilaments displaced to the periphery. This striking segregation precedes the abnormal and excessive neurofilament accumulation in these diseases, which in turn leads to focal axonal swellings. While neurofilament accumulation suggests an impairment of neurofilament transport along axons, the underlying mechanism of their segregation from microtubules remains poorly understood for over 30 years. To address this question, we developed a stochastic multiscale model for the cross-sectional distribution of microtubules and neurofilaments in axons. The model describes microtubules, neurofilaments and organelles as interacting particles in a 2D cross-section, and is built upon molecular processes that occur on a time scale of seconds or shorter. It incorporates the longitudinal transport of neurofilaments and organelles through this domain by allowing stochastic arrival and departure of these cargoes, and integrates the dynamic interactions of these cargoes with microtubules mediated by molecular motors. Simulations of the model demonstrate that organelles can pull nearby microtubules together, and in the absence of neurofilament transport, this mechanism gradually segregates microtubules from neurofilaments on a time scale of hours, similar to that observed in toxic neuropathies. This suggests that the microtubule-neurofilament segregation can be a consequence of the selective impairment of neurofilament transport. The model generates the experimentally testable

  7. A Stochastic Multiscale Model That Explains the Segregation of Axonal Microtubules and Neurofilaments in Neurological Diseases.

    PubMed

    Xue, Chuan; Shtylla, Blerta; Brown, Anthony

    2015-08-01

    The organization of the axonal cytoskeleton is a key determinant of the normal function of an axon, which is a long thin projection of a neuron. Under normal conditions two axonal cytoskeletal polymers, microtubules and neurofilaments, align longitudinally in axons and are interspersed in axonal cross-sections. However, in many neurotoxic and neurodegenerative disorders, microtubules and neurofilaments segregate apart from each other, with microtubules and membranous organelles clustered centrally and neurofilaments displaced to the periphery. This striking segregation precedes the abnormal and excessive neurofilament accumulation in these diseases, which in turn leads to focal axonal swellings. While neurofilament accumulation suggests an impairment of neurofilament transport along axons, the underlying mechanism of their segregation from microtubules remains poorly understood for over 30 years. To address this question, we developed a stochastic multiscale model for the cross-sectional distribution of microtubules and neurofilaments in axons. The model describes microtubules, neurofilaments and organelles as interacting particles in a 2D cross-section, and is built upon molecular processes that occur on a time scale of seconds or shorter. It incorporates the longitudinal transport of neurofilaments and organelles through this domain by allowing stochastic arrival and departure of these cargoes, and integrates the dynamic interactions of these cargoes with microtubules mediated by molecular motors. Simulations of the model demonstrate that organelles can pull nearby microtubules together, and in the absence of neurofilament transport, this mechanism gradually segregates microtubules from neurofilaments on a time scale of hours, similar to that observed in toxic neuropathies. This suggests that the microtubule-neurofilament segregation can be a consequence of the selective impairment of neurofilament transport. The model generates the experimentally testable

  8. Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins.

    PubMed

    Sanjuán Szklarz, Luiza K; Kozjak-Pavlovic, Vera; Vögtle, F-Nora; Chacinska, Agnieszka; Milenkovic, Dusanka; Vogel, Sandra; Dürr, Mark; Westermann, Benedikt; Guiard, Bernard; Martinou, Jean-Claude; Borner, Christoph; Pfanner, Nikolaus; Meisinger, Chris

    2007-04-20

    The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.

  9. The peroxisome proliferator perfluorodecanoic acid inhibits the peripheral-type benzodiazepine receptor (PBR) expression and hormone-stimulated mitochondrial cholesterol transport and steroid formation in Leydig cells.

    PubMed

    Boujrad, N; Vidic, B; Gazouli, M; Culty, M; Papadopoulos, V

    2000-09-01

    The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

  10. Compositional analysis of growing axons from rat sympathetic neurons

    PubMed Central

    1978-01-01

    We describe culture systems for neurons of an adrenergic autonomic ganglion which: (a) permit cultivation of neurons without supporting cells, (b) permit separate harvest of somal and axonal material, and (c) permit direct access to the neuronal surface. The antimetabolites used to suppress supporting cell growth did not have demonstrable effects on neuronal polypeptide synthesis. Rapid neurite outgrowth, which characterized these cultures, was prevented by colchicine or cycloheximide and resumed promptly after their withdrawal. Axons separated from cell bodies showed no incorporation of label from leucine or fucose, but did exhibit incorporation of glucosamine. The major polypeptides present in this neuron, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, are described. No major differences in polypeptide content were observed when soma and axons were compared. Likewise, there were no differences detected in polypeptides synthesized by neurons in suspension or neurons actively extending processes. Analysis of the polypeptides within the neurites after labeling with amino acids indicated transport at a number of different rates; certain of these polypeptides corresponded in size and transport characteristics to polypeptides observed in the rabbit optic nerve after labeling of retinal ganglion cells. Tubulin and actin have been definitively identified in this cell type (18); we found proteins similar in size and proportionate amounts to be among the rapidly transported soluble polypeptides. The prominent polypeptides observed after several methods of surface labeling are described. PMID:151689

  11. An Organelle Gatekeeper Function for Caenorhabditis elegans UNC-16 (JIP3) at the Axon Initial Segment

    PubMed Central

    Edwards, Stacey L.; Yu, Szi-chieh; Hoover, Christopher M.; Phillips, Barret C.; Richmond, Janet E.; Miller, Kenneth G.

    2013-01-01

    Neurons must cope with extreme membrane trafficking demands to produce axons with organelle compositions that differ dramatically from those of the cell soma and dendrites; however, the mechanism by which they accomplish this is not understood. Here we use electron microscopy and quantitative imaging of tagged organelles to show that Caenorhabditis elegans axons lacking UNC-16 (JIP3/Sunday Driver) accumulate Golgi, endosomes, and lysosomes at levels up to 10-fold higher than wild type, while ER membranes are largely unaffected. Time lapse microscopy of tagged lysosomes in living animals and an analysis of lysosome distributions in various regions of unc-16 mutant axons revealed that UNC-16 inhibits organelles from escaping the axon initial segment (AIS) and moving to the distal synaptic part of the axon. Immunostaining of native UNC-16 in C. elegans neurons revealed a localized concentration of UNC-16 at the initial segment, although UNC-16 is also sparsely distributed in distal regions of axons, including the synaptic region. Organelles that escape the AIS in unc-16 mutants show bidirectional active transport within the axon commissure that occasionally deposits them in the synaptic region, where their mobility decreases and they accumulate. These results argue against the long-standing, untested hypothesis that JIP3/Sunday Driver promotes anterograde organelle transport in axons and instead suggest an organelle gatekeeper model in which UNC-16 (JIP3/Sunday Driver) selectively inhibits the escape of Golgi and endosomal organelles from the AIS. This is the first evidence for an organelle gatekeeper function at the AIS, which could provide a regulatory node for controlling axon organelle composition. PMID:23633144

  12. Antibody-Mediated Oligodendrocyte Remyelination Promotes Axon Health in Progressive Demyelinating Disease.

    PubMed

    Wootla, Bharath; Denic, Aleksandar; Watzlawik, Jens O; Warrington, Arthur E; Rodriguez, Moses

    2016-10-01

    Demyelination underlies early neurological symptoms in multiple sclerosis (MS); however, axonal damage is considered critical for permanent chronic deficits. The precise mechanisms by which axonal injury occurs in MS are unclear; one hypothesis is the absence or failure of remyelination, suggesting that promoting remyelination may protect axons from death. This report provides direct evidence that promoting oligodendrocyte remyelination protects axons and maintains transport function. Persistent Theiler's virus infection of Swiss Jim Lambert (SJL)/J mice was used as a model of MS to assess the effects of remyelination on axonal injury following demyelination in the spinal cord. Remyelination was induced using an oligodendrocyte/myelin-specific recombinant human monoclonal IgM, rHIgM22. The antibody is endowed with strong anti-apoptotic and pro-proliferative effects on oligodendrocyte progenitor cells. We used (1)H-magnetic resonance spectroscopy (MRS) at the brainstem to measure N-acetyl-aspartate (NAA) as a surrogate of neuronal health and spinal cord integrity. We found increased brainstem NAA concentrations at 5 weeks post-treatment with rHIgM22, which remained stable out to 10 weeks. Detailed spinal cord morphology studies revealed enhanced remyelination in the rHIgM22-treated group but not in the isotype control antibody- or saline-treated groups. Importantly, we found rHIgM22-mediated remyelination protected small- and medium-caliber mid-thoracic spinal cord axons from damage despite similar demyelination and inflammation across all experimental groups. The most direct confirmation of remyelination-mediated protection of descending neurons was an improvement in retrograde transport. Treatment with rHIgM22 significantly increased the number of retrograde-labeled neurons in the brainstem, indicating that preserved axons are functionally competent. This is direct validation that remyelination preserves spinal cord axons and protects functional axon integrity

  13. Salicylic acid binding of mitochondrial alpha-ketoglutarate dehydrogenase E2 affects mitochondrial oxidative phosphorylation and electron transport chain components and plays a role in basal defense against tobacco mosaic virus in tomato.

    PubMed

    Liao, Yangwenke; Tian, Miaoying; Zhang, Huan; Li, Xin; Wang, Yu; Xia, Xiaojian; Zhou, Jie; Zhou, Yanhong; Yu, Jingquan; Shi, Kai; Klessig, Daniel F

    2015-02-01

    Salicylic acid (SA) plays a critical role in plant defense against pathogen invasion. SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense and involves a specific pathway mediated by mitochondria; however, the underlying mechanisms remain largely unknown. The SA-binding activity of the recombinant tomato (Solanum lycopersicum) alpha-ketoglutarate dehydrogenase (Slα-kGDH) E2 subunit of the tricarboxylic acid (TCA) cycle was characterized. The biological role of this binding in plant defenses against tobacco mosaic virus (TMV) was further investigated via Slα-kGDH E2 silencing and transient overexpression in plants. Slα-kGDH E2 was found to bind SA in two independent assays. SA treatment, as well as Slα-kGDH E2 silencing, increased resistance to TMV. SA did not further enhance TMV defense in Slα-kGDH E2-silenced tomato plants but did reduce TMV susceptibility in Nicotiana benthamiana plants transiently overexpressing Slα-kGDH E2. Furthermore, Slα-kGDH E2-silencing-induced TMV resistance was fully blocked by bongkrekic acid application and alternative oxidase 1a silencing. These results indicated that binding by Slα-kGDH E2 of SA acts upstream of and affects the mitochondrial electron transport chain, which plays an important role in basal defense against TMV. The findings of this study help to elucidate the mechanisms of SA-induced viral defense.

  14. Formation and Regulation of Mitochondrial Membranes

    PubMed Central

    Schenkel, Laila Cigana

    2014-01-01

    Mitochondrial membrane phospholipids are essential for the mitochondrial architecture, the activity of respiratory proteins, and the transport of proteins into the mitochondria. The accumulation of phospholipids within mitochondria depends on a coordinate synthesis, degradation, and trafficking of phospholipids between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. Several studies highlight the contribution of dietary fatty acids to the remodeling of phospholipids and mitochondrial membrane homeostasis. Understanding the role of phospholipids in the mitochondrial membrane and their metabolism will shed light on the molecular mechanisms involved in the regulation of mitochondrial function and in the mitochondrial-related diseases. PMID:24578708

  15. Precursor and mature NGF live tracking: one versus many at a time in the axons

    PubMed Central

    De Nadai, Teresa; Marchetti, Laura; Di Rienzo, Carmine; Calvello, Mariantonietta; Signore, Giovanni; Di Matteo, Pierluigi; Gobbo, Francesco; Turturro, Sabrina; Meucci, Sandro; Viegi, Alessandro; Beltram, Fabio; Luin, Stefano; Cattaneo, Antonino

    2016-01-01

    The classical view of nerve growth factor (NGF) action in the nervous system is linked to its retrograde axonal transport. However, almost nothing is known on the trafficking properties of its unprocessed precursor proNGF, characterized by different and generally opposite biological functions with respect to its mature counterpart. Here we developed a strategy to fluorolabel both purified precursor and mature neurotrophins (NTs) with a controlled stoichiometry and insertion site. Using a single particle tracking approach, we characterized the axonal transport of proNGF versus mature NGF in living dorsal root ganglion neurons grown in compartmentalized microfluidic devices. We demonstrate that proNGF is retrogradely transported as NGF, but with a lower flux and a different distribution of numbers of neurotrophins per vesicle. Moreover, exploiting a dual-color labelling technique, we analysed the transport of both NT forms when simultaneously administered to the axon tips. PMID:26829890

  16. Local translation and directional steering in axons

    PubMed Central

    Lin, Andrew C; Holt, Christine E

    2007-01-01

    The assembly of functional neural circuits in the developing brain requires neurons to extend axons to the correct targets. This in turn requires the navigating tips of axons to respond appropriately to guidance cues present along the axonal pathway, despite being cellular ‘outposts' far from the soma. Work over the past few years has demonstrated a critical role for local translation within the axon in this process in vitro, making axon guidance another process that requires spatially localized translation, among others such as synaptic plasticity, cell migration, and cell polarity. This article reviews recent findings in local axonal translation and discusses how new protein synthesis may function in growth cone guidance, with a comparative view toward models of local translation in other systems. PMID:17660744

  17. Degeneration and regeneration of ganglion cell axons.

    PubMed

    Weise, J; Ankerhold, R; Bähr, M

    2000-01-15

    The retino-tectal system has been used to study developmental aspects of axon growth, synapse formation and the establishment of a precise topographic order as well as degeneration and regeneration of adult retinal ganglion cell (RGC) axons after axonal lesion. This paper reviews some novel findings that provide new insights into the mechanisms of developmental RGC axon growth, pathfinding, and target formation. It also focuses on the cellular and molecular cascades that underlie RGC degeneration following an axonal lesion and on some therapeutic strategies to enhance survival of axotomized RGCs in vivo. In addition, this review deals with problems related to the induction of regeneration after axonal lesion in the adult CNS using the retino-tectal system as model. Different therapeutic approaches to promote RGC regeneration and requirements for specific target formation of regenerating RGCs in vitro and in vivo are discussed. PMID:10649506

  18. Axonal Localization of Neuritin/CPG15 mRNA in Neuronal Populations through Distinct 5′ and 3′ UTR Elements

    PubMed Central

    Merianda, Tanuja T.; Gomes, Cynthia; Yoo, Soonmoon; Vuppalanchi, Deepika

    2013-01-01

    Many neuronal mRNAs are actively transported into distal axons. The 3′ untranslated regions (UTRs) of axonal mRNAs often contain cues for their localization. The 3′ UTR of neuritin mRNA was shown to be sufficient for localization into axons of hippocampal neurons. Here, we show that neuritin mRNA localizes into axons of rat sensory neurons, but this is predominantly driven by the 5′ rather than 3′ UTR. Neuritin mRNA shifts from cell body to axon predominantly after nerve crush injury, suggesting that it encodes a growth-associated protein. Consistent with this, overexpression of neuritin increases axon growth but only when its mRNA localizes into the axons. PMID:23966695

  19. PROTECTIVE EFFECTS OF POTASSIUM TRANSPORT IN MITOCHONDRIA FROM RAT MYOMETRIUM UNDER ACTIVATION OF MITOCHONDRIAL PERMEABILITY TRANSITION PORE.

    PubMed

    Vadzyuk, O B

    2015-01-01

    We demonstrated using PBFI K(+)-sensitive fluorescent probe an enhancement of both components of K(+)-cycle--the ATP-sensitive K(+)-uptake and quinine-sensitive K+/H(+)-exchange--under the Ca2+ induced opening of mitochondrial permeability transition pore (MPTP) in rat myometrium mitochondria. Addition of CaCl2 (100 μM to K(+)-free medium results in the enhancement of reactive oxygen species (ROS) production, which was eliminated by cyclosporine A. Addition of CaCl2 to K(+)-rich medium did not increase the rate of ROS production, but blocking of mitoK+(ATP)-channels with glybenclamide (10 mcM increased production of ROS. We conclude that K(+)-cycle exerts a protective influence in mitochondria from rat myometrium by regulation of matrix volume and rate of ROS production under the condition of Ca(2+)-induced MPTP.

  20. Axonal Targeting of Trk Receptors via Transcytosis Regulates Sensitivity to Neurotrophin Responses

    PubMed Central

    Ascaño, Maria; Richmond, Alissa; Borden, Philip; Kuruvilla, Rejji

    2009-01-01

    Axonal targeting of trophic receptors is critical for neuronal responses to extracellular developmental cues, yet the underlying trafficking mechanisms remain unclear. Here, we report that Trk receptors for target-derived neurotrophins are anterogradely trafficked to axons via transcytosis in sympathetic neurons. Using compartmentalized cultures, we show that mature receptors on neuronal soma surfaces are endocytosed and remobilized via Rab11-positive recycling endosomes into axons. Inhibition of dynamin-dependent endocytosis disrupted anterograde transport and localization of TrkA receptors in axons. Anterograde TrkA delivery and exocytosis into axon growth cones is enhanced by nerve growth factor (NGF), acting locally on distal axons. Perturbing endocytic recycling attenuated NGF-dependent signaling and axon growth, while enhancing recycling conferred increased neuronal sensitivity to NGF. Our results reveal regulated transcytosis as an unexpected mode of Trk trafficking that serves to rapidly mobilize ready-synthesized receptors to growth cones, thus providing a positive feedback mechanism by which limiting concentrations of target-derived neurotrophins enhance neuronal sensitivity. PMID:19759314

  1. The use of proteomic analysis to study trafficking defects in axons.

    PubMed

    Fu, Xiaoqin; Brown, Kristy J; Rayavarapu, Sree; Nagaraju, Kanneboyina; Liu, Judy S

    2016-01-01

    Mutations in microtubule subunits and microtubule-associated proteins are the causes of many neurological disorders. These human conditions are usually associated with axonal tract defects or degeneration. The molecular mechanisms of these axonal dysfunction are still largely unknown. Conventional methods may not yield a complete analysis of downstream molecules related to axonal dysfunctions. Therefore, we devised a simple unbiased method to screen molecular motors and axonal molecules, which might be involved in axonal defects. We performed our analysis in the mouse with a targeted deletion in the doublecortin (Dcx) gene. Dcx is a microtubule-associated protein with direct effects on microtubule motors. Furthermore, the knockout of Dcx and its functionally redundant structurally similar paralog, doublecortin-like kinase 1 (Dclk1), in mouse results in thinner or absent axon tracts, including the corpus callosum and anterior commissures. We compared protein profiles of corpus callosum from Dcx knockout and wild-type mouse of P0-P2 using mass spectrometry. This strategy allowed us to identify novel candidates downstream of Dcx involved in axon transport.

  2. Retinal Afferent Ingrowth to Neocortical Transplants in the Adult Rat Superior Colliculus is due to the Regeneration of Damaged Axons

    PubMed Central

    Ross, D. T.; Das, G. D.

    1994-01-01

    Retinal afferent ingrowth to embryonic neural transplants in the adult rat superior colliculus may represent either sprouting of intact axons or the regeneration of transected axons. If ingrowth represents regeneration of damaged retinofugai axons, then lesions that axotomize more retinofugal axons at the transplantation site should induce greater retinal afferent ingrowth. Alternately, if ingrowth represents terminal or collateral sprouting of intact retinofugal axons at or near the transplant/host optic layer interface, then the magnitude of retinal afferent ingrowth should be directly related to the total area of this interface. To test between these two hypotheses surgical knife wounds were made either parallel (in the sagittal plane) or perpendicular (in the transverse plane) to the course of axons in the stratum opticum, embryonic neocortical tissue was transplanted at the coordinates of these tectal slits, and retinal afferent ingrowth visualized 1-90 days after surgery using anterogradely transported HRP. A zone of traumatic reaction (ztr) in the optic layers was seen in every case, characterized by hypertrophied axons and swollen terminal clubs at 1 day. Between 30 and 90 days the damaged retinofugal axons in the zone formed dense fascicles and neuroma-like tangles. Retinal afferent ingrowth occurred only across transplant interface regions with the ztr. The magnitude of ingrowth was directly related to the area of the ztr interface and not the total optic layer interface area. Retinal afferent ingrowth appears to reflect the intrinsic regenerative capacity of adult mammalian retinal ganglion cells and not sprouting of undamaged axons. PMID:7703292

  3. Mitochondrial vasculopathy

    PubMed Central

    Finsterer, Josef; Zarrouk-Mahjoub, Sinda

    2016-01-01

    Mitochondrial disorders (MIDs) are usually multisystem disorders (mitochondrial multiorgan disorder syndrome) either on from onset or starting at a point during the disease course. Most frequently affected tissues are those with a high oxygen demand such as the central nervous system, the muscle, endocrine glands, or the myocardium. Recently, it has been shown that rarely also the arteries may be affected (mitochondrial arteriopathy). This review focuses on the type, diagnosis, and treatment of mitochondrial vasculopathy in MID patients. A literature search using appropriate search terms was carried out. Mitochondrial vasculopathy manifests as either microangiopathy or macroangiopathy. Clinical manifestations of mitochondrial microangiopathy include leukoencephalopathy, migraine-like headache, stroke-like episodes, or peripheral retinopathy. Mitochondrial macroangiopathy manifests as atherosclerosis, ectasia of arteries, aneurysm formation, dissection, or spontaneous rupture of arteries. The diagnosis relies on the documentation and confirmation of the mitochondrial metabolic defect or the genetic cause after exclusion of non-MID causes. Treatment is not at variance compared to treatment of vasculopathy due to non-MID causes. Mitochondrial vasculopathy exists and manifests as micro- or macroangiopathy. Diagnosing mitochondrial vasculopathy is crucial since appropriate treatment may prevent from severe complications. PMID:27231520

  4. AxonSeg: Open Source Software for Axon and Myelin Segmentation and Morphometric Analysis

    PubMed Central

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

    2016-01-01

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

  5. AxonSeg: Open Source Software for Axon and Myelin Segmentation and Morphometric Analysis

    PubMed Central

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

    2016-01-01

    Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy (SEM) only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLAB-based graphical user interface (GUI) and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing; (ii) pre-segmentation of axons over a cropped image and discriminant analysis (DA) to select the best parameters based on axon shape and intensity information; (iii) automatic axon and myelin segmentation over the full image; and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), SEM and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology. AxonSeg is open source and freely available at: https://github.com/neuropoly/axonseg. PMID:27594833

  6. AxonSeg: Open Source Software for Axon and Myelin Segmentation and Morphometric Analysis.

    PubMed

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

    2016-01-01

    Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy (SEM) only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLAB-based graphical user interface (GUI) and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing; (ii) pre-segmentation of axons over a cropped image and discriminant analysis (DA) to select the best parameters based on axon shape and intensity information; (iii) automatic axon and myelin segmentation over the full image; and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), SEM and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology. AxonSeg is open source and freely available at: https://github.com/neuropoly/axonseg. PMID:27594833

  7. Effects of TAT-conjugated platinum nanoparticles on lifespan of mitochondrial electron transport complex I-deficient Caenorhabditis elegans, nuo-1.

    PubMed

    Sakaue, Yuri; Kim, Juewon; Miyamoto, Yusei

    2010-01-01

    Platinum nanoparticle (Pt-np) species are superoxide dismutase/catalase mimetics and also have an activity similar to that of mitochondrial electron transport complex I. To examine if this complex I-like activity functions in vivo, we studied the effects of Pt-nps on the lifespan of a mitochondrial complex I-deficient Caenorhabditis elegans mutant, nuo-1 (LB25) compared with wild-type N2. We synthesized a fusion protein of a cell-penetrating peptide, human immunodeficiency virus-1 TAT (48-60), C-terminally linked to a peptide with a high affinity to platinum (GRKKRRQRRRPPQ-DRTSTWR). Pt-nps were functionalized by conjugation with this fusion protein at a 1:1 ratio of TAT-PtBP to Pt atoms. Adult worms were treated with conjugated Pt-nps for 10 days. The mean lifespan of untreated N2 and LB25 was 19.6 ± 0.4 and 11.8 ± 0.3 days, respectively. Using 5 μM of conjugated Pt-nps, the lifespan of N2 and LB25 was maximally extended. This maximal lifespan extension of LB25 was 31.9 ± 2.6%, which was significantly greater than that of N2 (21.1 ± 1.7%, P < 0.05 by Student's t-test). Internalization of Pt into the whole body and mitochondria was similar between these two strains. Excessive accumulation of reactive oxygen species was not observed in the cytosol or mitochondria of untreated LB25. Treatment for five days with 5 μM conjugated Pt-nps decreased cytosolic and mitochondrial reactive oxygen species in N2 and LB25 to a similar extent. The ratio of [NAD(+)]/[NADH] was very low in the whole body and mitochondria of control LB25. After five days of treatment with 5 μM conjugated Pt-nps, the ratio of [NAD(+)]/[NADH] was increased in N2 and LB25. However, the degree of the increase was much higher in LB25 than in N2. Pt-nps function as NADH oxidase and recover the [NAD(+)]/[NADH] ratio in LB25, leading to effective extension of the lifespan of LB25. PMID:20957220

  8. Regulating Axonal Responses to Injury: The Intersection between Signaling Pathways Involved in Axon Myelination and The Inhibition of Axon Regeneration.

    PubMed

    Rao, Sudheendra N R; Pearse, Damien D

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  10. Effect of Ganoderma lucidum on the activities of mitochondrial dehydrogenases and complex I and II of electron transport chain in the brain of aged rats.

    PubMed

    Ajith, T A; Sudheesh, N P; Roshny, D; Abishek, G; Janardhanan, K K

    2009-03-01

    Dysfunction of the mitochondrial respiratory chain, being direct intracellular source of reactive oxygen species (ROS), is important in the pathogenesis of number of ageing associated human disorders. Effect of ethanol extract of Ganoderma lucidum on the activities of mitochondrial dehydrogenases; complex I and II of electron transport chain have been evaluated in the aged rat brain. Aged male Wistar rats were administered with ethanol extract of G. lucidum (50 and 250mg/kg, p.o) once daily for 15 days. Similarly DL-alpha-lipoic acid (100mg/kg, p.o) administered group was kept as the reference standard. Young and aged rats administered with water were kept as young and aged control, respectively. The effect of treatment was assessed by estimating the activities of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), alpha-ketoglutarate dehydrogenase (alpha-KGDH), pyruvate dehydrogenase (PDH), complex I and II in the mitochondria of rat brain. Results of the study demonstrated that the extract of G. lucidum (50 and 250mg/kg) significantly (p<0.01) enhanced the activities of PDH, alpha-KGDH, SDH, complex I and II when compared to that of the aged control animals. The level of the lipid peroxidation was significantly lowered (p<0.01) in the G. lucidum treated group with respect to that of aged control. However, we could not find any statistically significant difference between the activities of enzymes in groups treated with 50 and 250mg/kg of G. lucidum. The activity exhibited by the extract of G. lucidum in the present study can be partially correlated to its antioxidant activity. The results of the study concluded that the extract of G. lucidum may effective to improve the function of mitochondria in aged rat brain, suggest its possible therapeutic application against ageing associated neurodegenerative diseases. PMID:19041385

  11. Axonal interferon responses and alphaherpesvirus neuroinvasion

    NASA Astrophysics Data System (ADS)

    Song, Ren

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

  12. Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana.

    PubMed

    Cabassa-Hourton, Cécile; Schertl, Peter; Bordenave-Jacquemin, Marianne; Saadallah, Kaouthar; Guivarc'h, Anne; Lebreton, Sandrine; Planchais, Séverine; Klodmann, Jennifer; Eubel, Holger; Crilat, Emilie; Lefebvre-De Vos, Delphine; Ghelis, Thanos; Richard, Luc; Abdelly, Chedly; Carol, Pierre; Braun, Hans-Peter; Savouré, Arnould

    2016-09-01

    Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by MS. The 2D gels showed that ProDH1 forms part of a low-molecular-mass (70-140 kDa) complex in the mitochondrial membrane. To evaluate the contribution of each isoform to proline oxidation, mitochondria were isolated from wild-type (WT) and prodh1, prodh2, prodh1prodh2 and p5cdh mutants. ProDH activity was high for genotypes in which ProDH, most likely ProDH1, was strongly induced by proline. Respiratory measurements indicate that ProDH1 has a role in oxidizing excess proline and transferring electrons to the respiratory chain. PMID:27303048

  13. Uptake of nerve growth factor along peripheral and spinal axons of primary sensory neurons

    SciTech Connect

    Richardson, P.M.; Riopelle, R.J.

    1984-07-01

    To investigate the distribution of nerve growth factor (NGF) receptors on peripheral and central axons, (/sup 125/I)NGF was injected into the sciatic nerve or spinal cord of adult rats. Accumulation of (/sup 125/I)NGF in lumbar dorsal root ganglia was monitored by gamma emission counting and radioautography. (/sup 125/I)NGF, injected endoneurially in small quantities, was taken into sensory axons by a saturable process and was transported retrogradely to their cell bodies at a maximal rate of 2.5 to 7.5 mm/hr. Because very little (/sup 125/I)NGF reached peripheral terminals, the results were interpreted to indicate that receptors for NGF are present on nonterminal segments of sensory axons. The specificity and high affinity of NGF uptake were illustrated by observations that negligible amounts of gamma activity accumulated in lumbar dorsal root ganglia after comparable intraneural injection of (/sup 125/I) cytochrome C or (/sup 125/I)oxidized NGF. Similar techniques were used to