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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Chen, Yanmin

    2013-01-01

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

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

    PubMed

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

    2011-05-11

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

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

    PubMed

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

    2014-02-01

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

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

    PubMed

    Reddy, P Hemachandra; Shirendeb, Ulziibat P

    2012-02-01

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

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

    PubMed

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

    2012-01-15

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

  10. 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

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

    PubMed

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

    2011-12-01

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

  12. Increased mitochondrial content in remyelinated axons: implications for multiple sclerosis

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2014-04-23

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

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

    PubMed

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

    2017-07-01

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

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

    PubMed Central

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

    2014-01-01

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

  17. Calcium release from intra-axonal endoplasmic reticulum leads to axon degeneration through mitochondrial dysfunction.

    PubMed

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

    2014-05-21

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

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

    PubMed Central

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

    2014-01-01

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

  19. Cargo distributions differentiate pathological axonal transport impairments.

    PubMed

    Mitchell, Cassie S; Lee, Robert H

    2012-05-07

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

  20. 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

  1. Methodological advances in imaging intravital axonal transport.

    PubMed

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

    2017-01-01

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

  2. Methodological advances in imaging intravital axonal transport

    PubMed Central

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

    2017-01-01

    Axonal transport is the active process whereby neurons transport cargoes such as organelles and proteins anterogradely from the cell body to the axon terminal and retrogradely in the opposite direction. Bi-directional transport in axons is absolutely essential for the functioning and survival of neurons and appears to be negatively impacted by both aging and diseases of the nervous system, such as Alzheimer’s disease and amyotrophic lateral sclerosis. The movement of individual cargoes along axons has been studied in vitro in live neurons and tissue explants for a number of years; however, it is currently unclear as to whether these systems faithfully and consistently replicate the in vivo situation. A number of intravital techniques originally developed for studying diverse biological events have recently been adapted to monitor axonal transport in real-time in a range of live organisms and are providing novel insight into this dynamic process. Here, we highlight these methodological advances in intravital imaging of axonal transport, outlining key strengths and limitations while discussing findings, possible improvements, and outstanding questions. PMID:28344778

  3. Axonal Degeneration Is Mediated by the Mitochondrial Permeability Transition Pore

    PubMed Central

    Barrientos, Sebastian A.; Martinez, Nicolas W.; Yoo, Soonmoon; Jara, Juan S.; Zamorano, Sebastian; Hetz, Claudio; Twiss, Jeffery L.; Alvarez, Jaime; Court, Felipe A.

    2011-01-01

    Axonal degeneration is an active process that has been associated with neurodegenerative conditions triggered by mechanical, metabolic, infectious, toxic, hereditary and inflammatory stimuli. This degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. Several signaling pathways are implicated in axonal degeneration, but identification of an integrative mechanism for this self-destructive process has remained elusive. Here, we show that rapid axonal degeneration triggered by distinct mechanical and toxic insults is dependent on the activation of the mitochondrial permeability transition pore (mPTP). Both pharmacological and genetic targeting of cyclophilin D, a functional component of the mPTP, protects severed axons and vincristine-treated neurons from axonal degeneration in ex vivo and in vitro mouse and rat model systems. These effects were observed in axons from both the peripheral and central nervous system. Our results suggest that the mPTP is a key effector of axonal degeneration, upon which several independent signaling pathways converge. Since axonal and synapse degeneration are increasingly considered early pathological events in neurodegeneration, our work identifies a potential target for therapeutic intervention in a wide variety of conditions that lead to loss of axons and subsequent functional impairment. PMID:21248121

  4. Geniposide Alleviates Amyloid-Induced Synaptic Injury by Protecting Axonal Mitochondrial Trafficking

    PubMed Central

    Zhang, Haijing; Zhao, Chunhui; Lv, Cui; Liu, Xiaoli; Du, Shijing; Li, Zhi; Wang, Yongyan; Zhang, Wensheng

    2017-01-01

    Synaptic and mitochondrial pathologies are early events in the progression of Alzheimer's disease (AD). Normal axonal mitochondrial function and transport play crucial roles in maintaining synaptic function by producing high levels of adenosine triphosphate and buffering calcium. However, there can be abnormal axonal mitochondrial trafficking, distribution, and fragmentation, which are strongly correlated with amyloid-β (Aβ)-induced synaptic loss and dysfunction. The present study examined the neuroprotective effect of geniposide, a compound extracted from gardenia fruit in Aβ-treated neurons and an AD mouse model. Geniposide alleviated Aβ-induced axonal mitochondrial abnormalities by increasing axonal mitochondrial density and length and improving mitochondrial motility and trafficking in cultured hippocampal neurons, consequently ameliorating synaptic damage by reversing synaptic loss, addressing spine density and morphology abnormalities, and ameliorating the decreases in synapse-related proteins in neurons and APPswe/PS1dE9 mice. These findings provide new insights into the effects of geniposide administration on neuronal and synaptic functions under conditions of Aβ enrichment. PMID:28179878

  5. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-08-18

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

  8. 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

  9. 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

  10. Modelling organelle transport after traumatic axonal injury.

    PubMed

    Kuznetsov, I A; Kuznetsov, A V

    2015-01-01

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

  11. 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.

  12. The Interplay of Axonal Energy Homeostasis and Mitochondrial Trafficking and Anchoring.

    PubMed

    Sheng, Zu-Hang

    2017-02-19

    Mitochondria are key cellular power plants essential for neuronal growth, survival, function, and regeneration after injury. Given their unique morphological features, neurons face exceptional challenges in maintaining energy homeostasis at distal synapses and growth cones where energy is in high demand. Efficient regulation of mitochondrial trafficking and anchoring is critical for neurons to meet altered energy requirements. Mitochondrial dysfunction and impaired transport have been implicated in several major neurological disorders. Thus, research into energy-mediated regulation of mitochondrial recruitment and redistribution is an important emerging frontier. In this review, I discuss new insights into the mechanisms regulating mitochondrial trafficking and anchoring, and provide an updated overview of how mitochondrial motility maintains energy homeostasis in axons, thus contributing to neuronal growth, regeneration, and synaptic function.

  13. 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".

  14. 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

  15. Mitochondrial ABC transporters.

    PubMed

    Lill, R; Kispal, G

    2001-01-01

    In contrast to bacteria, mitochondria contain only a few ATP binding cassette (ABC) transporters in their inner membrane. The known mitochondrial ABC proteins fall into two major classes that, in the yeast Saccharomyces cerevisiae, are represented by the half-transporter Atm1p and the two closely homologous proteins Mdl1p and Mdl2p. In humans two Atm1p orthologues (ABC7 and MTABC3) and two proteins homologous to Mdll/2p have been localized to mitochondria. The Atm1p-like proteins perform an important function in mitochondrial iron homeostasis and in the maturation of Fe/S proteins in the cytosol. Mutations in ABC7 are causative of hereditary X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). MTABC3 may be a candidate gene for the lethal neonatal syndrome. The function of the mitochondrial Mdl1/2p-like proteins is not clear at present with the notable exception of murine ABC-me that may transport intermediates of heme biosynthesis from the matrix to the cytosol in erythroid tissues.

  16. Fast axonal transport in early experimental disc edema.

    PubMed

    Radius, R L; Anderson, D R

    1980-02-01

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

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

    PubMed Central

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

    2012-01-01

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

  18. 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

  19. 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.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2017-09-14

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

  3. Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

    PubMed

    Gale, Jenna R; Aschrafi, Armaz; Gioio, Anthony E; Kaplan, Barry B

    2017-06-01

    Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones. Accumulating evidence suggests that the local translation of these mRNAs is required for mitochondrial maintenance and axonal viability. Here, we review evidence that suggests a critical role for axonal translation of nuclear-encoded mitochondrial mRNAs in axonal growth and development. Additionally, we explore the role that site-specific translation at the mitochondria itself may play in this process. Finally, we briefly review the clinical implications of dysregulation of local translation of mitochondrial-related mRNAs in neurodevelopmental disorders.

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

    PubMed

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

    2012-06-20

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

    1993-01-01

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

  7. Cytoplasmic dynein is associated with slow axonal transport.

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

  10. 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).

  11. Morphology of axonal transport abnormalities in primate eyes.

    PubMed

    Radius, R L; Anderson, D R

    1981-11-01

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

  12. Morphology of axonal transport abnormalities in primate eyes.

    PubMed Central

    Radius, R L; Anderson, D R

    1981-01-01

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

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

    PubMed

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

    2008-06-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-08

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

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

    PubMed

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

    2016-01-01

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

  18. Mitochondrial biogenesis and fission in axons in cell culture and animal models of diabetic neuropathy.

    PubMed

    Vincent, Andrea M; Edwards, James L; McLean, Lisa L; Hong, Yu; Cerri, Federica; Lopez, Ignazio; Quattrini, Angelo; Feldman, Eva L

    2010-10-01

    Mitochondrial-mediated oxidative stress in response to high glucose is proposed as a primary cause of dorsal root ganglia (DRG) neuron injury in the pathogenesis of diabetic neuropathy. In the present study, we report a greater number of mitochondria in both myelinated and unmyelinated dorsal root axons in a well-established model of murine diabetic neuropathy. No similar changes were seen in younger diabetic animals without neuropathy or in the ventral motor roots of any diabetic animals. These findings led us to examine mitochondrial biogenesis and fission in response to hyperglycemia in the neurites of cultured DRG neurons. We demonstrate overall mitochondrial biogenesis via increases in mitochondrial transcription factors and increases in mitochondrial DNA in both DRG neurons and axons. However, this process occurs over a longer time period than a rapidly observed increase in the number of mitochondria in DRG neurites that appears to result, at least in part, from mitochondrial fission. We conclude that during acute hyperglycemia, mitochondrial fission is a prominent response, and excessive mitochondrial fission may result in dysregulation of energy production, activation of caspase 3, and subsequent DRG neuron injury. During more prolonged hyperglycemia, there is evidence of compensatory mitochondrial biogenesis in axons. Our data suggest that an imbalance between mitochondrial biogenesis and fission may play a role in the pathogenesis of diabetic neuropathy.

  19. 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.

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

    PubMed

    Bergquist, J E; Hanson, M

    1983-03-01

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

  1. Increased axonal mitochondrial activity as an adaptation to myelin deficiency in the Shiverer mouse.

    PubMed

    Andrews, Helen; White, Kathryn; Thomson, Christine; Edgar, Julia; Bates, David; Griffiths, Ian; Turnbull, Douglass; Nichols, Philip

    2006-06-01

    Axonal pathology in multiple sclerosis (MS) has been described for over a century, but new insights into axonal loss and disability have refocused interest in this area. There is evidence of oxidative damage to mitochondrial DNA in chronic MS plaques, suggesting that mitochondrial failure may play a role in MS pathology. We propose that in the chronic absence of myelin the maintenance of conduction relies partially on an increase in mitochondria to provide energy. This increased energy requirement also promotes reactive oxygen species (ROS), because most intraaxonal ROS are generated by mitochondria. If antioxidant defenses are overwhelmed by an excess of ROS, this may result in damage to the axon. Our aim was to investigate whether a chronic lack of myelin results in adaptive changes involving mitochondria within the axon. We investigated this in the shiverer mouse. This myelin basic protein gene mutant provides a model of how adult central nervous system (CNS) axons cope with the chronic absence of a compact myelin sheath. Cytochrome c histochemistry demonstrated a twofold increase in mitochondrial activity in white matter tracts of shiverer, and electron microscopy confirmed a significantly higher number of mitochondria within the dysmyelinated axons. Our data demonstrate that there are adaptive changes involving mitochondria occurring within CNS axons in shiverer mice in response to a lack of myelin. This work contributes to our understanding of the adaptive changes occurring in response to a lack of myelin in a noninflammatory environment similar to the situation seen in chronically demyelinated MS plaques.

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

    PubMed Central

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

    2012-01-01

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

  3. Mechanistic logic underlying the axonal transport of cytosolic proteins

    PubMed Central

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

    2011-01-01

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

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

    PubMed

    Minoshima, Satoshi; Cross, Donna

    2008-03-01

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

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

    PubMed

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

    1986-01-01

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

  6. 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

  7. Loss of Mitochondrial Fission Depletes Axonal Mitochondria in Midbrain Dopamine Neurons

    PubMed Central

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

    2014-01-01

    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. PMID:25339743

  8. 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.

  9. 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.

  10. Mitochondrial dysfunction and oxidative damage cooperatively fuel axonal degeneration in X-linked adrenoleukodystrophy.

    PubMed

    Fourcade, Stéphane; López-Erauskin, Jone; Ruiz, Montserrat; Ferrer, Isidre; Pujol, Aurora

    2014-03-01

    X-linked adrenoleukodystrophy (X-ALD) is the most frequent inherited monogenic demyelinating disease (minimal incidence 1:17,000). It is often lethal and currently lacks a satisfactory therapy. The disease is caused by loss of function of the ABCD1 gene, a peroxisomal ATP-binding cassette transporter, resulting in the accumulation of VLCFA (very long-chain fatty acids) in organs and plasma. Understanding of the aetiopathogenesis is a prerequisite for the development of novel therapeutic strategies. Functional genomics analysis of an ABCD1 null mouse, a mouse model for adrenomyeloneuropathy, has revealed presymptomatic alterations in several metabolic pathways converging on redox and bioenergetic homeostasis, with failure of mitochondrial OXPHOS disruption and mitochondrial depletion. These defects could be major contributors to the neurodegenerative cascade, as has been reported in several neurodegenerative disorders. Drugs targeting the redox imbalance/mitochondria dysfunction interplay have shown efficacy at halting axonal degeneration and associated disability in the mouse, and thus offer therapeutic hope. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

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

    PubMed

    Weiss, Kurt R; Littleton, J Troy

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

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

    PubMed

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

    1994-11-01

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

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

    SciTech Connect

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

    1983-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  15. Aberrant Schwann cell lipid metabolism linked to mitochondrial deficits leads to axon degeneration and neuropathy.

    PubMed

    Viader, Andreu; Sasaki, Yo; Kim, Sungsu; Strickland, Amy; Workman, Cayce S; Yang, Kui; Gross, Richard W; Milbrandt, Jeffrey

    2013-03-06

    Mitochondrial dysfunction is a common cause of peripheral neuropathy. Much effort has been devoted to examining the role played by neuronal/axonal mitochondria, but how mitochondrial deficits in peripheral nerve glia (Schwann cells [SCs]) contribute to peripheral nerve diseases remains unclear. Here, we investigate a mouse model of peripheral neuropathy secondary to SC mitochondrial dysfunction (Tfam-SCKOs). We show that disruption of SC mitochondria activates a maladaptive integrated stress response (ISR) through the actions of heme-regulated inhibitor (HRI) kinase, and causes a shift in lipid metabolism away from fatty acid synthesis toward oxidation. These alterations in SC lipid metabolism result in depletion of important myelin lipid components as well as in accumulation of acylcarnitines (ACs), an intermediate of fatty acid β-oxidation. Importantly, we show that ACs are released from SCs and induce axonal degeneration. A maladaptive ISR as well as altered SC lipid metabolism are thus underlying pathological mechanisms in mitochondria-related peripheral neuropathies.

  16. 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

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

    PubMed Central

    1981-01-01

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

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

    PubMed

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

    2009-11-09

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

  19. 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

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

    PubMed

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

    2016-03-22

    Redox switches are important mediators in neoplastic, cardiovascular and neurological disorders. We recently identified spontaneous redox signals in neurons at the single mitochondrion level where transients of glutathione oxidation go along with shortening and re-elongation of the organelle. We now have developed advanced image and signal-processing methods to re-assess and extend previously obtained data. Here we analyze redox and pH signals of entire mitochondrial populations. In total, we quantified the effects of 628 redox and pH events in 1797 mitochondria from intercostal axons and neuromuscular synapses using optical sensors (mito-Grx1-roGFP2; mito-SypHer). We show that neuronal mitochondria can undergo multiple redox cycles exhibiting markedly different signal characteristics compared to single redox events. Redox and pH events occur more often in mitochondrial clusters (medium cluster size: 34.1 ± 4.8 μm(2)). Local clusters possess higher mitochondrial densities than the rest of the axon, suggesting morphological and functional inter-mitochondrial coupling. We find that cluster formation is redox sensitive and can be blocked by the antioxidant MitoQ. In a nerve crush paradigm, mitochondrial clusters form sequentially adjacent to the lesion site and oxidation spreads between mitochondria. Our methodology combines optical bioenergetics and advanced signal processing and allows quantitative assessment of entire mitochondrial populations.

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

    PubMed

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

    1998-04-20

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

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

    PubMed

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

    1977-12-19

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

  3. 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

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

    PubMed

    Radius, R L; Anderson, D R

    1981-04-01

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

  5. Age-Related Changes in Axonal and Mitochondrial Ultrastructure and Function in White Matter

    PubMed Central

    Stahon, Katharine E.; Bastian, Chinthasagar; Griffith, Shelby; Kidd, Grahame J.; Brunet, Sylvain

    2016-01-01

    The impact of aging on CNS white matter (WM) is of general interest because the global effects of aging on myelinated nerve fibers are more complex and profound than those in cortical gray matter. It is important to distinguish between axonal changes created by normal aging and those caused by neurodegenerative diseases, including multiple sclerosis, stroke, glaucoma, Alzheimer's disease, and traumatic brain injury. Using three-dimensional electron microscopy, we show that in mouse optic nerve, which is a pure and fully myelinated WM tract, aging axons are larger, have thicker myelin, and are characterized by longer and thicker mitochondria, which are associated with altered levels of mitochondrial shaping proteins. These structural alterations in aging mitochondria correlate with lower ATP levels and increased generation of nitric oxide, protein nitration, and lipid peroxidation. Moreover, mitochondria–smooth endoplasmic reticulum interactions are compromised due to decreased associations and decreased levels of calnexin and calreticulin, suggesting a disruption in Ca2+ homeostasis and defective unfolded protein responses in aging axons. Despite these age-related modifications, axon function is sustained in aging WM, which suggests that age-dependent changes do not lead to irreversible functional decline under normal conditions, as is observed in neurodegenerative diseases. SIGNIFICANCE STATEMENT Aging is a common risk factor for a number of neurodegenerative diseases, including stroke. Mitochondrial dysfunction and oxidative damage with age are hypothesized to increase risk for stroke. We compared axon–myelin–node–mitochondrion–smooth endoplasmic reticulum (SER) interactions in white matter obtained at 1 and 12 months. We show that aging axons have enlarged volume, thicker myelin, and elongated and thicker mitochondria. Furthermore, there are reduced SER connections to mitochondria that correlate with lower calnexin and calreticulin levels. Despite a

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

    PubMed Central

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

    2012-01-01

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

  7. 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

  8. 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.

  9. Effect of kinesin velocity distribution on slow axonal transport

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Andrey

    2012-08-01

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

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

    PubMed

    Sadananda, Aparna; Ray, Krishanu

    2012-09-01

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

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

    PubMed

    Radius, R L; Bade, B

    1982-10-01

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

  12. 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.

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

    PubMed Central

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

    2012-01-01

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

  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

    2011-07-06

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

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

    PubMed

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

    2006-05-01

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

  16. Mitochondrial dynamics and peripheral neuropathy.

    PubMed

    Baloh, Robert H

    2008-02-01

    Peripheral neuropathy is perhaps the archetypal disease of axonal degeneration, characteristically involving degeneration of the longest axons in the body. Evidence from both inherited and acquired forms of peripheral neuropathy strongly supports that the primary pathology is in the axons themselves and points to disruption of axonal transport as an important disease mechanism. Recent studies in human genetics have further identified abnormalities in mitochondrial dynamics--the fusion, fission, and movement of mitochondria--as a player in the pathogenesis of inherited peripheral neuropathy. This review provides an update on the mechanisms of mitochondrial trafficking in axons and the emerging relationship between the disruption of mitochondrial dynamics and axonal degeneration. Evidence suggests mitochondria are a "critical cargo" whose transport is necessary for proper axonal and synaptic function. Importantly, understanding the regulation of mitochondrial movement and the consequences of decreased axonal mitochondrial function may define new paths for therapeutic agents in peripheral neuropathy and other neurodegenerative diseases.

  17. 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

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

    SciTech Connect

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

    1989-03-01

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

  19. Age-Related Changes in Axonal and Mitochondrial Ultrastructure and Function in White Matter.

    PubMed

    Stahon, Katharine E; Bastian, Chinthasagar; Griffith, Shelby; Kidd, Grahame J; Brunet, Sylvain; Baltan, Selva

    2016-09-28

    The impact of aging on CNS white matter (WM) is of general interest because the global effects of aging on myelinated nerve fibers are more complex and profound than those in cortical gray matter. It is important to distinguish between axonal changes created by normal aging and those caused by neurodegenerative diseases, including multiple sclerosis, stroke, glaucoma, Alzheimer's disease, and traumatic brain injury. Using three-dimensional electron microscopy, we show that in mouse optic nerve, which is a pure and fully myelinated WM tract, aging axons are larger, have thicker myelin, and are characterized by longer and thicker mitochondria, which are associated with altered levels of mitochondrial shaping proteins. These structural alterations in aging mitochondria correlate with lower ATP levels and increased generation of nitric oxide, protein nitration, and lipid peroxidation. Moreover, mitochondria-smooth endoplasmic reticulum interactions are compromised due to decreased associations and decreased levels of calnexin and calreticulin, suggesting a disruption in Ca(2+) homeostasis and defective unfolded protein responses in aging axons. Despite these age-related modifications, axon function is sustained in aging WM, which suggests that age-dependent changes do not lead to irreversible functional decline under normal conditions, as is observed in neurodegenerative diseases. Aging is a common risk factor for a number of neurodegenerative diseases, including stroke. Mitochondrial dysfunction and oxidative damage with age are hypothesized to increase risk for stroke. We compared axon-myelin-node-mitochondrion-smooth endoplasmic reticulum (SER) interactions in white matter obtained at 1 and 12 months. We show that aging axons have enlarged volume, thicker myelin, and elongated and thicker mitochondria. Furthermore, there are reduced SER connections to mitochondria that correlate with lower calnexin and calreticulin levels. Despite a prominent decrease in number

  20. Mitochondrial DNA double-strand breaks in oligodendrocytes cause demyelination, axonal injury and CNS inflammation.

    PubMed

    Madsen, Pernille M; Pinto, Milena; Patel, Shreyans; McCarthy, Stephanie; Gao, Han; Taherian, Mehran; Karmally, Shaffiat; Pereira, Claudia V; Dvoriantchikova, Galina; Ivanov, Dmitry; Tanaka, Kenji F; Moraes, Carlos T; Brambilla, Roberta

    2017-09-20

    Mitochondrial dysfunction has been implicated in the pathophysiology of neurodegenerative disorders, including multiple sclerosis (MS). To date, the investigation of mitochondrial dysfunction in MS has focused exclusively on neurons, with no studies exploring whether dysregulation of mitochondrial bioenergetics and/or genetics in oligodendrocytes might be associated with the etiopathogenesis of MS and other demyelinating syndromes. To address this question, we established a mouse model where mitochondrial DNA (mtDNA) double-strand breaks (DSB) were specifically induced in myelinating oligodendrocytes (PLP:mtPstI mice) by expressing a mitochondrial-targeted endonuclease, mtPstI, starting at 3 weeks of age. In both female and male mice, DSB of oligodendroglial mtDNA caused impairment of locomotor function, chronic demyelination, glial activation and axonal degeneration, which became more severe with time of induction. In addition, after short transient induction of mtDNA DSB, PLP:mtPstI mice showed an exacerbated response to experimental autoimmune encephalomyelitis. Together, our data demonstrate that mtDNA damage can cause primary oligodendropathy which in turn triggers demyelination, proving PLP:mtPstI mice to be a useful tool to study the pathological consequences of mitochondrial dysfunction in oligodendrocytes. In addition, the demyelination and axonal loss displayed by PLP:mtPstI mice recapitulate some of the key features of chronic demyelinating syndromes, including progressive MS forms, which are not accurately reproduced in the models currently available. For this reason the PLP:mtPstI mouse represents a unique and much needed platform for testing remyelinating therapies.SIGNIFICANCE STATEMENTMadsen et al. show that oligodendrocyte-specific mtDNA double strand breaks in PLP:mtPstI mice cause oligodendrocyte death and demyelination associated with axonal damage and glial activation. Hence, PLP:mtPstI mice represent a unique tool to study the pathological

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

    PubMed

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

    2014-11-01

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

  2. 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

  3. News and views on mitochondrial water transport.

    PubMed

    Gena, Patrizia; Fanelli, Elena; Brenner, Catherine; Svelto, Maria; Calamita, Giuseppe

    2009-01-01

    The osmotic movement of water into and out of the mitochondrial matrix underlies the extraordinary plasticity that characterizes mitochondria, a feature of pivotal importance to cell bioenergetics and signaling, and of critical relevance to life-and-death cell decision. However, the biophysics and identity of mitochondrial water transport had remained mostly unexplored, until recent works suggesting high water permeability and the presence of multiple facilitated pathways of water diffusion in liver mitochondria. Here, we attempt to summarize our current view of the mechanisms of mitochondrial water transport and possible relevance of the channel-mediated pathways created by mitochondrial permeability transition, aquaporins and protein/lipid specializations. Assessing the molecular bases and dynamics of mitochondrial water permeability will help to answer the much-debated question over the role of mitochondria.

  4. 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

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

    PubMed

    McQuarrie, I G

    1986-12-01

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

  6. Abnormal mitochondrial transport and morphology as early pathological changes in human models of spinal muscular atrophy

    PubMed Central

    Xu, Chong-Chong; Denton, Kyle R.; Wang, Zhi-Bo; Zhang, Xiaoqing; Li, Xue-Jun

    2016-01-01

    ABSTRACT Spinal muscular atrophy (SMA), characterized by specific degeneration of spinal motor neurons, is caused by mutations in the survival of motor neuron 1, telomeric (SMN1) gene and subsequent decreased levels of functional SMN. How the deficiency of SMN, a ubiquitously expressed protein, leads to spinal motor neuron-specific degeneration in individuals affected by SMA remains unknown. In this study, we examined the role of SMN in mitochondrial axonal transport and morphology in human motor neurons by generating SMA type 1 patient-specific induced pluripotent stem cells (iPSCs) and differentiating these cells into spinal motor neurons. The initial specification of spinal motor neurons was not affected, but these SMA spinal motor neurons specifically degenerated following long-term culture. Moreover, at an early stage in SMA spinal motor neurons, but not in SMA forebrain neurons, the number of mitochondria, mitochondrial area and mitochondrial transport were significantly reduced in axons. Knocking down of SMN expression led to similar mitochondrial defects in spinal motor neurons derived from human embryonic stem cells, confirming that SMN deficiency results in impaired mitochondrial dynamics. Finally, the application of N-acetylcysteine (NAC) mitigated the impairment in mitochondrial transport and morphology and rescued motor neuron degeneration in SMA long-term cultures. Furthermore, NAC ameliorated the reduction in mitochondrial membrane potential in SMA spinal motor neurons, suggesting that NAC might rescue apoptosis and motor neuron degeneration by improving mitochondrial health. Overall, our data demonstrate that SMN deficiency results in abnormal mitochondrial transport and morphology and a subsequent reduction in mitochondrial health, which are implicated in the specific degeneration of spinal motor neurons in SMA. PMID:26586529

  7. Abnormal mitochondrial transport and morphology as early pathological changes in human models of spinal muscular atrophy.

    PubMed

    Xu, Chong-Chong; Denton, Kyle R; Wang, Zhi-Bo; Zhang, Xiaoqing; Li, Xue-Jun

    2016-01-01

    Spinal muscular atrophy (SMA), characterized by specific degeneration of spinal motor neurons, is caused by mutations in the survival of motor neuron 1, telomeric (SMN1) gene and subsequent decreased levels of functional SMN. How the deficiency of SMN, a ubiquitously expressed protein, leads to spinal motor neuron-specific degeneration in individuals affected by SMA remains unknown. In this study, we examined the role of SMN in mitochondrial axonal transport and morphology in human motor neurons by generating SMA type 1 patient-specific induced pluripotent stem cells (iPSCs) and differentiating these cells into spinal motor neurons. The initial specification of spinal motor neurons was not affected, but these SMA spinal motor neurons specifically degenerated following long-term culture. Moreover, at an early stage in SMA spinal motor neurons, but not in SMA forebrain neurons, the number of mitochondria, mitochondrial area and mitochondrial transport were significantly reduced in axons. Knocking down of SMN expression led to similar mitochondrial defects in spinal motor neurons derived from human embryonic stem cells, confirming that SMN deficiency results in impaired mitochondrial dynamics. Finally, the application of N-acetylcysteine (NAC) mitigated the impairment in mitochondrial transport and morphology and rescued motor neuron degeneration in SMA long-term cultures. Furthermore, NAC ameliorated the reduction in mitochondrial membrane potential in SMA spinal motor neurons, suggesting that NAC might rescue apoptosis and motor neuron degeneration by improving mitochondrial health. Overall, our data demonstrate that SMN deficiency results in abnormal mitochondrial transport and morphology and a subsequent reduction in mitochondrial health, which are implicated in the specific degeneration of spinal motor neurons in SMA.

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

    PubMed Central

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

    2009-01-01

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

  9. Oxidative stress, mitochondrial and proteostasis malfunction in adrenoleukodystrophy: A paradigm for axonal degeneration.

    PubMed

    Fourcade, Stéphane; Ferrer, Isidre; Pujol, Aurora

    2015-11-01

    Peroxisomal and mitochondrial malfunction, which are highly intertwined through redox regulation, in combination with defective proteostasis, are hallmarks of the most prevalent multifactorial neurodegenerative diseases-including Alzheimer's (AD) and Parkinson's disease (PD)-and of the aging process, and are also found in inherited conditions. Here we review the interplay between oxidative stress and axonal degeneration, taking as groundwork recent findings on pathomechanisms of the peroxisomal neurometabolic disease adrenoleukodystrophy (X-ALD). We explore the impact of chronic redox imbalance caused by the excess of very long-chain fatty acids (VLCFA) on mitochondrial respiration and biogenesis, and discuss how this impairs protein quality control mechanisms essential for neural cell survival, such as the proteasome and autophagy systems. As consequence, prime molecular targets in the pathogenetic cascade emerge, such as the SIRT1/PGC-1α axis of mitochondrial biogenesis, and the inhibitor of autophagy mTOR. Thus, we propose that mitochondria-targeted antioxidants; mitochondrial biogenesis boosters such as the antidiabetic pioglitazone and the SIRT1 ligand resveratrol; and the autophagy activator temsirolimus, a derivative of the mTOR inhibitor rapamycin, hold promise as disease-modifying therapies for X-ALD. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

  11. 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.

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

    PubMed Central

    1980-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2012-08-15

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Tasdemir-Yilmaz, Ozge E; Segal, Rosalind A

    2016-08-01

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

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

    PubMed

    Lund, L M; McQuarrie, I G

    2001-12-21

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

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

    PubMed Central

    Petersen, Jennifer D.; Kaech, Stefanie

    2014-01-01

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

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

    DTIC Science & Technology

    2015-10-01

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

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

    PubMed Central

    Jakobsen, J; Sidenius, P; Braendgaard, H

    1986-01-01

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

  1. Mitochondrial swelling impairs the transport of organelles in cerebellar granule neurons.

    PubMed

    Kaasik, Allen; Safiulina, Dzhamilja; Choubey, Vinay; Kuum, Malle; Zharkovsky, Alexander; Veksler, Vladimir

    2007-11-09

    Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.

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

    PubMed

    Radius, R L

    1980-11-01

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

  3. 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.

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

    PubMed Central

    1980-01-01

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

  5. Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models.

    PubMed

    Magrané, Jordi; Cortez, Czrina; Gan, Wen-Biao; Manfredi, Giovanni

    2014-03-15

    Neuronal mitochondrial morphology abnormalities occur in models of familial amyotrophic lateral sclerosis (ALS) associated with SOD1 and TDP43 mutations. These abnormalities have been linked to mitochondrial axonal transport defects, but the temporal and spatial relationship between mitochondrial morphology and transport alterations in these two distinct genetic forms of ALS has not been investigated in vivo. To address this question, we crossed SOD1 (wild-type SOD1(WT) and mutant SOD1(G93A)) or TDP43 (mutant TDP43(A315T)) transgenic mice with mice expressing the fluorescent protein Dendra targeted to mitochondria in neurons (mitoDendra). At different time points during the disease course, we studied mitochondrial transport in the intact sciatic nerve of living mice and analyzed axonal mitochondrial morphology at multiple sites, spanning from the spinal cord to the motor terminals. Defects of retrograde mitochondrial transport were detected at 45 days of age, before the onset of symptoms, in SOD1(G93A) and TDP43(A315T) mice, but not in SOD1(WT). At later disease stages, also anterograde mitochondrial transport was affected in both mutant mouse lines. In SOD1(G93A) mice, mitochondrial morphological abnormalities were apparent at 15 days of age, thus preceding transport abnormalities. Conversely, in TDP43(A315T) mice, morphological abnormalities appeared after the onset of transport defects. Taken together, these findings demonstrate that neuronal mitochondrial transport and morphology abnormalities occur in vivo and that they are common denominators of different genetic forms of the ALS. At the same time, differences in the temporal and spatial manifestation of mitochondrial abnormalities between the two mouse models of familial ALS imply that different molecular mechanisms may be involved.

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

    PubMed

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

    2013-01-31

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-03-20

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

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

    PubMed

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

    2017-09-17

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

    Naftelberg, Shiran; Ast, Gil; Perlson, Eran

    2017-01-01

    Familial dysautonomia (FD) is a rare children neurodegenerative disease caused due to a point mutation in the IKBKAP gene that results in decreased IKK complex-associated protein (IKAP) protein production. The disease affects mostly the dorsal root ganglion (DRG) and the sympathetic ganglion. Recently, we found that the molecular mechanisms underlying neurodegeneration in FD patients are defects in axonal transport of nerve growth factors and microtubule stability in the DRG. Neurons are highly polarized cells with very long axons. In order to survive and maintain proper function, neurons depend on transport of proteins and other cellular components from the neuronal body along the axons. We further demonstrated that IKAP is necessary for axon maintenance and showed that phosphatidylserine acts as an HDAC6 inhibitor to rescue neuronal function in FD cells. In this review, we will highlight our latest research findings. PMID:28553323

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

    PubMed

    Naftelberg, Shiran; Ast, Gil; Perlson, Eran

    2017-04-01

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

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

    PubMed

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

    2012-12-01

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2010-11-01

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

  18. 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.

  19. 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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

    1982-01-01

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

  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. 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

  3. 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

  4. Mitochondrial abnormality in sensory, but not motor, axons in paclitaxel-evoked painful peripheral neuropathy in the rat.

    PubMed

    Xiao, W H; Zheng, H; Zheng, F Y; Nuydens, R; Meert, T F; Bennett, G J

    2011-12-29

    The dose-limiting side effect of the anti-neoplastic agent, paclitaxel, is a chronic distal symmetrical peripheral neuropathy that produces sensory dysfunction (hypoesthesia and neuropathic pain) but little or no distal motor dysfunction. Similar peripheral neuropathies are seen with chemotherapeutics in the vinca alkaloid, platinum-complex, and proteasome inhibitor classes. Studies in rats suggest that the cause is a mitotoxic effect on axonal mitochondria. If so, then the absence of motor dysfunction may be due to mitotoxicity that affects sensory axons but spares motor axons. To investigate this, paclitaxel exposure levels in the dorsal root, ventral root, dorsal root ganglion, peripheral nerve, and spinal cord were measured, and the ultrastructure and the respiratory function of mitochondria in dorsal roots and ventral roots were compared. Sensory and motor axons in the roots and nerve had comparably low exposure to paclitaxel and exposure in the spinal cord was negligible. However, sensory neurons in the dorsal root ganglion had a very high and remarkably persistent (up to 10 days or more after the last injection) exposure to paclitaxel. Paclitaxel evoked a significant increase in the incidence of swollen and vacuolated mitochondria in the myelinated and unmyelinated sensory axons of the dorsal root (as seen previously in the peripheral nerve) but not in the motor axons of the ventral root. Stimulated mitochondrial respiration in the dorsal root was significantly depressed in paclitaxel-treated animals examined 2-4 weeks after the last injection, whereas respiration in the ventral root was normal. We conclude that the absence of motor dysfunction in paclitaxel-evoked peripheral neuropathy may be due to the absence of a mitotoxic effect in motor neuron axons, whereas the sensory dysfunction may be due to a mitotoxic effect resulting from the primary afferent neuron's cell body being exposed to high and persistent levels of paclitaxel.

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

    PubMed

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

    2009-05-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2015-07-15

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

  8. 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.

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

    PubMed Central

    Vagnoni, Alessio; Bullock, Simon L.

    2016-01-01

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

  10. 4-Hydroxy-2-nonenal induces mitochondrial dysfunction and aberrant axonal outgrowth in adult sensory neurons that mimics features of diabetic neuropathy.

    PubMed

    Akude, Eli; Zherebitskaya, Elena; Roy Chowdhury, Subir K; Girling, Kimberly; Fernyhough, Paul

    2010-01-01

    Modification of proteins by 4-hydroxy-2-nonenal (4-HNE) has been proposed to cause neurotoxicity in a number of neurodegenerative diseases, including distal axonopathy in diabetic sensory neuropathy. We tested the hypothesis that exposure of cultured adult rat sensory neurons to 4-HNE would result in the formation of amino acid adducts on mitochondrial proteins and that this process would be associated with impaired mitochondrial function and axonal regeneration. In addition, we compared 4-HNE-induced axon pathology with that exhibited by neurons isolated from diabetic rats. Cultured adult rat dorsal root ganglion (DRG) sensory neurons were incubated with varying concentrations of 4-HNE. Cell survival, axonal morphology, and level of axon outgrowth were assessed. In addition, video microscopy of live cells, western blot, and immunofluorescent staining were utilized to detect protein adduct formation by 4-HNE and to localize actively respiring mitochondria. 4-HNE induced formation of protein adducts on cytoskeletal and mitochondrial proteins, and impaired axon regeneration by approximately 50% at 3 microM while having no effect on neuronal survival. 4-HNE initiated formation of aberrant axonal structures and caused the accumulation of mitochondria in these dystrophic structures. Neurons treated with 4-HNE exhibited a distal loss of active mitochondria. Finally, the distal axonopathy and the associated aberrant axonal structures generated by 4-HNE treatment mimicked axon pathology observed in DRG sensory neurons isolated from diabetic rats and replicated aspects of neurodegeneration observed in human diabetic sensory neuropathy.

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

    PubMed

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

    2010-11-24

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

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

    PubMed

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

    2016-01-01

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

  13. 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…

  14. 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…

  15. 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

  16. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Gornstein, Erica L; Schwarz, Thomas L

    2017-02-01

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

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

    PubMed Central

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

    2016-01-01

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

  1. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2012-07-01

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

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

    PubMed

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

    2010-04-01

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

  5. 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

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

    SciTech Connect

    Crescitelli, L.A.

    1985-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  8. Mitochondrial lipid transport and biosynthesis: A complex balance

    PubMed Central

    2016-01-01

    Little is known about how mitochondrial lipids reach inner membrane–localized metabolic enzymes for phosphatidylethanolamine synthesis. Aaltonen et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201602007) and Miyata et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601082) now report roles for two mitochondrial complexes, Ups2–Mdm35 and mitochondrial contact site and cristae organizing system, in the biosynthesis and transport of mitochondrial lipids. PMID:27354376

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

    PubMed

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

    2015-11-20

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

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

    PubMed Central

    1981-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. 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.

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

    PubMed

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

    1999-05-06

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

  15. 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.

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

    PubMed

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

    2017-09-01

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

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

    SciTech Connect

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

    2005-05-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2016-12-01

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

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

    PubMed Central

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

    2016-01-01

    Familial Dysautonomia (FD) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO) mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs) with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF). These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6) levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration. PMID:27997532

  1. 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.

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Johnson, Christopher M.

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

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

    PubMed

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

    2010-01-01

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

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

    PubMed Central

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

    2012-01-01

    Summary Endocytosis of amyloid-β precursor protein (APP) is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP. PMID:23213470

  7. 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

  8. Mitochondrial motility in axons: membranous organelles may interact with the force generating system through multiple surface binding sites.

    PubMed

    Martz, D; Lasek, R J; Brady, S T; Allen, R D

    1984-01-01

    In living tissue, membrane-bound organelles, including mitochondria, move along parallel cytoplasmic pathways. Motion is directed and tends to be confined to a single path. Deviations from this single path motion are rare. When present, however, they tend to occur at points of intersection of cytoskeletal linear elements (LE). Such intersections are relatively uncommon in intact axons and extruded axoplasm. However, we have found that such intersections can be produced in extruded preparations by shear forces directed tangential to the axoplasmic surface. We have studied the detailed behavior of mitochondria in extruded squid axoplasm. Special attention was directed to the relationship between mitochondrial shape changes and orientation of cytoskeletal LE. The most striking of these changes in shape is branching. In this process, the mitochondrion transiently assumes a triradial (three-ended) shape. This appearance may be maintained for seconds to minutes before the normal cylindrical shape is resumed by absorption of either the newly formed end or, more commonly, one of the original ends. The frequency of branching appears to be dependent on the degree of cytoskeletal organization. It becomes more common as the number of apparent intersections between cytoskeletal LE increases. Further, the formation of new ends seems to occur along paths defined by cytoskeletal elements. These observations suggest that the mitochondrial membrane is multivalent. That is, it contains multiple sites capable of interacting with the axonal force generation apparatus. Furthermore, LE in the cytoskeleton may indicate the paths along which these interactions are permissible.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

    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

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

    DTIC Science & Technology

    2008-03-01

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

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

    PubMed

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

    2016-11-11

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

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

    PubMed

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

    2017-01-04

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

  15. 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.

  16. The role of mitochondrial transport in energy metabolism.

    PubMed

    Passarella, Salvatore; Atlante, Anna; Valenti, Daniela; de Bari, Lidia

    2003-04-01

    Since mitochondria are closed spaces in the cell, metabolite traffic across the mitochondrial membrane is needed to accomplish energy metabolism. The mitochondrial carriers play this function by uniport, symport and antiport processes. We give here a survey of about 50 transport processes catalysed by more than 30 carriers with a survey of the methods used to investigate metabolite transport in isolated mammalian mitochondria. The role of mitochondria in metabolic pathways including ammoniogenesis, amino acid metabolism, mitochondrial shuttles etc. is also reported in more detail, mainly in the light of the existence of new transport processes.

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

    PubMed Central

    1985-01-01

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

  18. Pathophysiology of mitochondrial volume homeostasis: potassium transport and permeability transition.

    PubMed

    Nowikovsky, Karin; Schweyen, Rudolf J; Bernardi, Paolo

    2009-05-01

    Regulation of mitochondrial volume is a key issue in cellular pathophysiology. Mitochondrial volume and shape changes can occur following regulated fission-fusion events, which are modulated by a complex network of cytosolic and mitochondrial proteins; and through regulation of ion transport across the inner membrane. In this review we will cover mitochondrial volume homeostasis that depends on (i) monovalent cation transport across the inner membrane, a regulated process that couples electrophoretic K(+) influx on K(+) channels to K(+) extrusion through the K(+)-H(+) exchanger; (ii) the permeability transition, a loss of inner membrane permeability that may be instrumental in triggering cell death. Specific emphasis will be placed on molecular advances on the nature of the transport protein(s) involved, and/or on diseases that depend on mitochondrial volume dysregulation.

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

    PubMed Central

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

    2013-01-01

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

  20. 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

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

    PubMed

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

    2006-03-01

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

  2. 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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Radius, R L; Bade, B

    1981-12-01

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

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

    PubMed Central

    Sheng, Zu-Hang; Cai, Qian

    2016-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2006-11-22

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    1994-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. 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

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

    PubMed

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

    2005-10-01

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

  15. The rice mitochondrial iron transporter is essential for plant growth

    PubMed Central

    Bashir, Khurram; Ishimaru, Yasuhiro; Shimo, Hugo; Nagasaka, Seiji; Fujimoto, Masaru; Takanashi, Hideki; Tsutsumi, Nobuhiro; An, Gynheung; Nakanishi, Hiromi; Nishizawa, Naoko K.

    2011-01-01

    In plants, iron (Fe) is essential for mitochondrial electron transport, heme, and Fe-Sulphur (Fe-S) cluster synthesis; however, plant mitochondrial Fe transporters have not been identified. Here we show, identify and characterize the rice mitochondrial Fe transporter (MIT). Based on a transfer DNA library screen, we identified a rice line showing symptoms of Fe deficiency while accumulating high shoot levels of Fe. Homozygous knockout of MIT in this line resulted in a lethal phenotype. MIT localized to the mitochondria and complemented the growth of Δmrs3Δmrs4 yeast defective in mitochondrial Fe transport. The growth of MIT-knockdown (mit-2) plants was also significantly impaired despite abundant Fe accumulation. Further, the decrease in the activity of the mitochondrial and cytosolic Fe-S enzyme, aconitase, indicated that Fe-S cluster synthesis is affected in mit-2 plants. These results indicate that MIT is a mitochondrial Fe transporter essential for rice growth and development. PMID:21610725

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

    Pandey, Jai P; Smith, Deanna S

    2011-01-01

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

  19. Automated measurement of fast mitochondrial transport in neurons.

    PubMed

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

    2015-01-01

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

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

    PubMed

    Lund, Linda M; McQuarrie, Irvine G

    2002-03-15

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

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

    PubMed Central

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

    2010-01-01

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

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

    DTIC Science & Technology

    2008-07-01

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

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

    PubMed Central

    Schwab, ME; Suda, K; Thoenen, H

    1979-01-01

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

  4. Dopamine transporter SPECT in patients with mitochondrial disorders

    PubMed Central

    Minnerop, M; Kornblum, C; Joe, A; Tatsch, K; Kunz, W; Klockgether, T; Wullner, U; Reinhardt, M

    2005-01-01

    Objective : To investigate the dopaminergic system in patients with known mitochondrial disorders and complex I deficiency. Methods: Dopamine transporter density was studied in 10 female patients with mitochondrial complex I deficiency by 123I-FP-CIT (N-ß-fluoropropyl-2ß-carbomethyl-3ß-(4-iodophenyl)-nortropane) SPECT. Results: No differences in 123I-FP-CIT striatal binding ratios were observed and no correlation of the degree of complex I deficiency and striatal binding ratios could be detected. Conclusions: These data argue against the possibility that mitochondrial complex I deficiency by itself is sufficient to elicit dopaminergic cell loss. PMID:15608010

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

    PubMed Central

    1987-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-10-01

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

  8. Local Translation of Extranuclear Lamin B Promotes Axon Maintenance

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

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

    1982-02-01

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

  10. Teaching the role of mitochondrial transport in energy metabolism.

    PubMed

    Passarella, Salvatore; Atlante, Anna

    2007-03-01

    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 metabolism has been investigated under a variety of physio-pathological conditions. Consistently we describe the procedure to investigate mitochondrial traffic in isolated mitochondria as a model system for students to learn. Here we report some observations that contribute to novel knowledge of the role of mitochondria in glycolysis, urea and purine nucleotide cycle, and nitrogen metabolism with particular reference to the malate/oxaloacetate shuttle and fumarate, glutamine, and lactate metabolism. Copyright © 2007 International Union of Biochemistry and Molecular Biology, Inc.

  11. 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.

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

    PubMed Central

    1983-01-01

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

  13. 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.

  14. 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

  15. 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

  16. 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

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

    PubMed

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

    2012-10-01

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

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

    PubMed

    Magen, Iddo; Gozes, Illana

    2013-12-01

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

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

    PubMed

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

    2003-08-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-20

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

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

    PubMed Central

    Kohler, H. -H.

    1977-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-14

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

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

    PubMed

    Hilton, Brett J; Bradke, Frank

    2017-10-01

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

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

    PubMed

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

    1980-02-01

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

  8. 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.

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

    PubMed

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

    2009-09-15

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

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

    PubMed

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

    2011-10-01

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

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

    PubMed

    Radius, R L

    1983-03-01

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

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

    PubMed

    Krench, Megan; Littleton, J Troy

    2013-01-01

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

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

    PubMed

    Edström, A; Sjöberg, J; Kanje, M

    1986-03-01

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

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

    PubMed

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

    2015-09-14

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

  15. 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

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

    PubMed

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

    2012-04-15

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

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

    PubMed

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

    2016-08-04

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

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

    PubMed Central

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

    2014-01-01

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

  19. 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

  20. Expression and putative role of mitochondrial transport proteins in cancer.

    PubMed

    Lytovchenko, Oleksandr; Kunji, Edmund R S

    2017-03-22

    Cancer cells undergo major changes in energy and biosynthetic metabolism. One of them is the Warburg effect, in which pyruvate is used for fermentation rather for oxidative phosphorylation. Another major one is their increased reliance on glutamine, which helps to replenish the pool of Krebs cycle metabolites used for other purposes, such as amino acid or lipid biosynthesis. Mitochondria are central to these alterations, as the biochemical pathways linking these processes run through these organelles. Two membranes, an outer and inner membrane, surround mitochondria, the latter being impermeable to most organic compounds. Therefore, a large number of transport proteins are needed to link the biochemical pathways of the cytosol and mitochondrial matrix. Since the transport steps are relatively slow, it is expected that many of these transport steps are altered when cells become cancerous. In this review, changes in expression and regulation of these transport proteins are discussed as well as the role of the transported substrates.

  1. [Molecular genetic studies of mitochondrial ornithine transporter deficiency (HHH syndrome)].

    PubMed

    Tsujino, S; Miyamoto, T; Kanazawa, N

    2001-11-01

    Mitochondrial ornithine transporter deficiency has been called HHH syndrome, because this disorder is characterized by three biochemical abnormalities; hyperornithinemia, hyperammonemia, and homocitrullinuria, and presents with various neurological symptoms; mental retardation, spastic paraparesis with pyramidal signs, cerebellar ataxia and episodic disturbance of consciousness or coma due to hyperammonemia. We identified four mutations in the mitochondrial ornithine transporter gene (ORNT1) of Japanese patients with HHH syndrome. These include a nonsense mutation (R179X), associated with exon skipping, missense mutations (G27E, P126R), and an insertion of AAC between codons 228 and 229, leading to an insertion of amino acid Asn. Especially, R179X was detected 4 of 7 Japanese patients (8 of 14 alleles), implying that this is a common mutation in Japanese population.

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

    PubMed

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

    1998-10-01

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

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

    SciTech Connect

    Maier, C.E.

    1989-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    1989-02-01

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

  6. [32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo.

    PubMed

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

    1994-11-01

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

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

    PubMed Central

    Lancaster, Karen Z.; Pfeiffer, Julie K.

    2010-01-01

    Poliovirus is an enteric virus that rarely invades the human central nervous system (CNS). To identify barriers limiting poliovirus spread from the periphery to CNS, we monitored trafficking of 10 marked viruses. After oral inoculation of susceptible mice, poliovirus was present in peripheral neurons, including vagus and sciatic nerves. To model viral trafficking in peripheral neurons, we intramuscularly injected mice with poliovirus, which follows a muscle–sciatic nerve–spinal cord–brain route. Only 20% of the poliovirus population successfully moved from muscle to brain, and three barriers limiting viral trafficking were identified. First, using light-sensitive viruses, we found limited viral replication in peripheral neurons. Second, retrograde axonal transport of poliovirus in peripheral neurons was inefficient; however, the efficiency was increased upon muscle damage, which also increased the transport efficiency of a non-viral neural tracer, wheat germ agglutinin. Third, using susceptible interferon (IFN) α/β receptor knockout mice, we demonstrated that the IFN response limited viral movement from the periphery to the brain. Surprisingly, the retrograde axonal transport barrier was equivalent in strength to the IFN barrier. Illustrating the importance of barriers created by the IFN response and inefficient axonal transport, IFN α/β receptor knockout mice with muscle damage permitted 80% of the viral population to access the brain, and succumbed to disease three times faster than mice with intact barriers. These results suggest that multiple separate barriers limit poliovirus trafficking from peripheral neurons to the CNS, possibly explaining the rare incidence of paralytic poliomyelitis. This study identifies inefficient axonal transport as a substantial barrier to poliovirus trafficking in peripheral neurons, which may limit CNS access for other viruses. PMID:20221252

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

    PubMed

    Lancaster, Karen Z; Pfeiffer, Julie K

    2010-03-05

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

  9. 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.

  10. 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.

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

    PubMed

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

    2016-03-01

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

  12. 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

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

    PubMed Central

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

    2000-01-01

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

  14. 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

  15. Removing dysfunctional mitochondria from axons independent of mitophagy under pathophysiological conditions.

    PubMed

    Lin, Mei-Yao; Cheng, Xiu-Tang; Xie, Yuxiang; Cai, Qian; Sheng, Zu-Hang

    2017-10-03

    Chronic mitochondrial dysfunction has been implicated in major neurodegenerative diseases. Long-term cumulative pathological stress leads to axonal accumulation of damaged mitochondria. Therefore, the early removal of defective mitochondria from axons constitutes a critical step of mitochondrial quality control. We recently investigated the axonal mitochondrial response to mild stress in wild-type neurons and chronic mitochondrial defects in amyotrophic lateral sclerosis (ALS)- and Alzheimer disease (AD)-linked neurons. We demonstrated that remobilizing stressed mitochondria is critical for maintaining axonal mitochondrial integrity. The selective release of the mitochondrial anchoring protein SNPH (syntaphilin) from stressed mitochondria enhances their retrograde transport toward the soma before PARK2/Parkin-mediated mitophagy is activated. This SNPH-mediated response is robustly activated during the early disease stages of ALS-linked motor neurons and AD-related cortical neurons. Our study thus reveals a new mechanism for the maintenance of axonal mitochondrial integrity through SNPH-mediated coordination of mitochondrial stress and motility that is independent of mitophagy.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    1995-06-01

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

  18. 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.

  19. 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

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

    PubMed

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

    1986-01-22

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

  1. HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation

    PubMed Central

    Hong, Young Bin; Choi, Heesun; Kim, Jisoo; Choi, Hyunjung; Mook-Jung, Inhee; Ha, Nina; Kyung, Jangbeen; Koo, Soo Kyung

    2016-01-01

    The Charcot-Marie-Tooth disease 2F (CMT2F) and distal hereditary motor neuropathy 2B (dHMN2B) are caused by autosomal dominantly inherited mutations of the heat shock 27 kDa protein 1 (HSPB1) gene and there are no specific therapies available yet. Here, we assessed the potential therapeutic effect of HDAC6 inhibitors on peripheral neuropathy with HSPB1 mutation using in vitro model of motor neurons derived from induced pluripotent stem cells (iPSCs) of CMT2F and dHMN2B patients. The absolute velocity of mitochondrial movements and the percentage of moving mitochondria in axons were lower both in CMT2F-motor neurons and in dHMN2B-motor neurons than those in controls, and the severity of the defective mitochondrial movement was different between the two disease models. CMT2F-motor neurons and dHMN2B-motor neurons also showed reduced α-tubulin acetylation compared with controls. The newly developed HDAC6 inhibitors, CHEMICAL X4 and CHEMICAL X9, increased acetylation of α-tubulin and reversed axonal movement defects of mitochondria in CMT2F-motor neurons and dHMN2B-motor neurons. Our results suggest that the neurons derived from patient-specific iPSCs can be used in drug screening including HDAC6 inhibitors targeting peripheral neuropathy. PMID:28105056

  2. HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation.

    PubMed

    Kim, Ji-Yon; Woo, So-Youn; Hong, Young Bin; Choi, Heesun; Kim, Jisoo; Choi, Hyunjung; Mook-Jung, Inhee; Ha, Nina; Kyung, Jangbeen; Koo, Soo Kyung; Jung, Sung-Chul; Choi, Byung-Ok

    2016-01-01

    The Charcot-Marie-Tooth disease 2F (CMT2F) and distal hereditary motor neuropathy 2B (dHMN2B) are caused by autosomal dominantly inherited mutations of the heat shock 27 kDa protein 1 (HSPB1) gene and there are no specific therapies available yet. Here, we assessed the potential therapeutic effect of HDAC6 inhibitors on peripheral neuropathy with HSPB1 mutation using in vitro model of motor neurons derived from induced pluripotent stem cells (iPSCs) of CMT2F and dHMN2B patients. The absolute velocity of mitochondrial movements and the percentage of moving mitochondria in axons were lower both in CMT2F-motor neurons and in dHMN2B-motor neurons than those in controls, and the severity of the defective mitochondrial movement was different between the two disease models. CMT2F-motor neurons and dHMN2B-motor neurons also showed reduced α-tubulin acetylation compared with controls. The newly developed HDAC6 inhibitors, CHEMICAL X4 and CHEMICAL X9, increased acetylation of α-tubulin and reversed axonal movement defects of mitochondria in CMT2F-motor neurons and dHMN2B-motor neurons. Our results suggest that the neurons derived from patient-specific iPSCs can be used in drug screening including HDAC6 inhibitors targeting peripheral neuropathy.

  3. 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

  4. 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.

  5. 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.

  6. 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

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

    PubMed Central

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

    2012-01-01

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

  8. Loss of MEC-17 Leads to Microtubule Instability and Axonal Degeneration

    PubMed Central

    Neumann, Brent; Hilliard, Massimo A.

    2014-01-01

    SUMMARY Axonal degeneration arises as a consequence of neuronal injury and is a common hallmark of a number of neurodegenerative diseases. However, the genetic causes and the cellular mechanisms that trigger this process are still largely unknown. Based on forward genetic screening in C. elegans, we have identified the α-tubulin acetyltransferase gene mec-17 as causing spontaneous, adult-onset, and progressive axonal degeneration. Loss of MEC-17 leads to microtubule instability, a reduction in mitochondrial number, and disrupted axonal transport, with altered distribution of both mitochondria and synaptic components. Furthermore, mec-17-mediated axonal degeneration occurs independently from its acetyltransferase domain; is enhanced by mutation of coel-1, a tubulin-associated molecule; and correlates with the animal’s body length. This study therefore identifies a critical role for the conserved microtubule-associated protein MEC-17 in preserving axon integrity and preventing axonal degeneration. PMID:24373971

  9. Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint.

    PubMed

    Yamamori, Tohru; Yasui, Hironobu; Yamazumi, Masayuki; Wada, Yusuke; Nakamura, Yoshinari; Nakamura, Hideo; Inanami, Osamu

    2012-07-15

    Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that

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

    PubMed

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

    2017-04-01

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

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

    PubMed

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

    1994-01-01

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

  12. Changes in mitochondrial electron transport chain activity during insect metamorphosis.

    PubMed

    Chamberlin, M E

    2007-02-01

    The midgut of the tobacco hornworm (Manduca sexta) is a highly aerobic tissue that is destroyed by programmed cell death during larval-pupal metamorphosis. The death of the epithelium begins after commitment to pupation, and the oxygen consumption of isolated midgut mitochondria decreases soon after commitment. To assess the role of the electron transport chain in this decline in mitochondrial function, the maximal activities of complexes I-IV of the respiratory chain were measured in isolated midgut mitochondria. Whereas there were no developmental changes in the activity of complex I or III, activities of complexes II and IV [cytochrome c oxidase (COX)] were higher in mitochondria from precommitment than postcommitment larvae. This finding is consistent with a higher rate of succinate oxidation in mitochondria isolated from precommitment larvae and reveals that the metamorphic decline in mitochondrial respiration is due to the targeted destruction or inactivation of specific sites within the mitochondria, rather than the indiscriminate destruction of the organelles. The COX turnover number (e- x s(-1) x cytochrome aa3(-1)) was greater for the enzyme from precommitment than postcommitment larvae, indicating a change in the enzyme structure and/or its lipid environment during the early stages of metamorphosis. The turnover number of COX in the intact mitochondria (in organello COX) was also lower in postcommitment larvae. In addition to changes in the protein or membrane phospholipids, the metamorphic decline in this rate constant may be a result of the observed loss of endogenous cytochrome c.

  13. 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

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

    PubMed Central

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

    2015-01-01

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

  15. Mitochondrial dynamics and inherited peripheral nerve diseases.

    PubMed

    Pareyson, Davide; Saveri, Paola; Sagnelli, Anna; Piscosquito, Giuseppe

    2015-06-02

    Peripheral nerves have peculiar energetic requirements because of considerable length of axons and therefore correct mitochondria functioning and distribution along nerves is fundamental. Mitochondrial dynamics refers to the continuous change in size, shape, and position of mitochondria within cells. Abnormalities of mitochondrial dynamics produced by mutations in proteins involved in mitochondrial fusion (mitofusin-2, MFN2), fission (ganglioside-induced differentiation-associated protein-1, GDAP1), and mitochondrial axonal transport usually present with a Charcot-Marie-Tooth disease (CMT) phenotype. MFN2 mutations cause CMT type 2A by altering mitochondrial fusion and trafficking along the axonal microtubule system. CMT2A is an axonal autosomal dominant CMT type which in most cases is characterized by early onset and rather severe course. GDAP1 mutations also alter fission, fusion and transport of mitochondria and are associated either with recessive demyelinating (CMT4A) and axonal CMT (AR-CMT2K) and, less commonly, with dominant, milder, axonal CMT (CMT2K). OPA1 (Optic Atrophy-1) is involved in fusion of mitochondrial inner membrane, and its heterozygous mutations lead to early-onset and progressive dominant optic atrophy which may be complicated by other neurological symptoms including peripheral neuropathy. Mutations in several proteins fundamental for the axonal transport or forming the axonal cytoskeleton result in peripheral neuropathy, i.e., CMT, distal hereditary motor neuropathy (dHMN) or hereditary sensory and autonomic neuropathy (HSAN), as well as in hereditary spastic paraplegia. Indeed, mitochondrial transport involves directly or indirectly components of the kinesin superfamily (KIF5A, KIF1A, KIF1B), responsible of anterograde transport, and of the dynein complex and related proteins (DYNC1H1, dynactin, dynamin-2), implicated in retrograde flow. Microtubules, neurofilaments, and chaperones such as heat shock proteins (HSPs) also have a fundamental

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

    PubMed

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

    1998-03-30

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

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

    PubMed Central

    1992-01-01

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

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

    PubMed

    Mollá, Belén; Muñoz-Lasso, Diana C; Riveiro, Fátima; Bolinches-Amorós, Arantxa; Pallardó, Federico V; Fernandez-Vilata, Angel; de la Iglesia-Vaya, María; Palau, Francesc; Gonzalez-Cabo, Pilar

    2017-01-01

    Friedreich's ataxia (FRDA) is a peripheral neuropathy involving a loss of proprioceptive sensory neurons. Studies of biopsies from patients suggest that axonal dysfunction precedes the death of proprioceptive neurons in a dying-back process. We observed that the deficiency of frataxin in sensory neurons of dorsal root ganglia (DRG) of the YG8R mouse model causes the formation of axonal spheroids which retain dysfunctional mitochondria, shows alterations in the cytoskeleton and it produces impairment of axonal transport and autophagic flux. The homogenous distribution of axonal spheroids along the neurites supports the existence of continues focal damages. This lead us to propose for FRDA a model of distal axonopathy based on axonal focal damages. In addition, we observed the involvement of oxidative stress and dyshomeostasis of calcium in axonal spheroid formation generating axonal injury as a primary cause of pathophysiology. Axonal spheroids may be a consequence of calcium imbalance, thus we propose the quenching or removal extracellular Ca(2+) to prevent spheroids formation. In our neuronal model, treatments with BAPTA and o-phenanthroline reverted the axonal dystrophy and the mitochondrial dysmorphic parameters. These results support the hypothesis that axonal pathology is reversible in FRDA by pharmacological manipulation of intracellular Ca(2+) with Ca(2+) chelators or metalloprotease inhibitors, preventing Ca(2+)-mediated axonal injury. Thus, the modulation of Ca(2+) levels may be a relevant therapeutic target to develop early axonal protection and prevent dying-back neurodegeneration.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. Mitochondrial Fusion/Fission, Transport and Autophagy in Parkinson's Disease: When Mitochondria Get Nasty

    PubMed Central

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

    2011-01-01

    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. PMID:21403911

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

    PubMed

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

    2013-10-18

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

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

    PubMed Central

    Forbes, Lindsey H.; Andrews, Melissa R.

    2017-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  8. Identification of Mitochondrial Coenzyme A Transporters from Maize and Arabidopsis1[W][OA

    PubMed Central

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

    2013-01-01

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

  9. A new mode of mitochondrial transport and polarized sorting regulated by Dynein, Milton and Miro.

    PubMed

    Melkov, Anna; Baskar, Raju; Alcalay, Yehonatan; Abdu, Uri

    2016-11-15

    Intrinsic cell microtubule (MT) polarity, together with molecular motors and adaptor proteins, determines mitochondrial polarized targeting and MT-dependent transport. In polarized cells, such as neurons, mitochondrial mobility and transport require the regulation of kinesin and dynein by two adaptor proteins, Milton and Miro. Recently, we found that dynein heavy chain 64C (Dhc64C) is the primary motor protein for both anterograde and retrograde transport of mitochondria in the Drosophila bristle. In this study, we show that a molecular lesion in the Dhc64C allele that reduced bristle mitochondrial velocity generated a variant that acts as a 'slow' dynein in an MT-gliding assay, indicating that dynein directly regulates mitochondrial transport. We also showed that in milton-RNAi flies, mitochondrial flux into the bristle shaft, but not velocity, was significantly reduced. Surprisingly, mitochondria retrograde flux, but not net velocity, was significantly decreased in miro-RNAi flies. We thus reveal a new mode of mitochondrial sorting in polarized cell growth, whereby bi-directional mitochondrial transport undertaken exclusively by dynein is regulated by Milton in the anterograde direction and by a Miro-dependent switch to the retrograde direction.

  10. 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.

  11. Mitochondrial pharmacology: electron transport chain bypass as strategies to treat mitochondrial dysfunction.

    PubMed

    Atamna, Hani; Mackey, Jeanette; Dhahbi, Joseph M

    2012-01-01

    Mitochondrial dysfunction (primary or secondary) is detrimental to intermediary metabolism. Therapeutic strategies to treat/prevent mitochondrial dysfunction could be valuable for managing metabolic and age-related disorders. Here, we review strategies proposed to treat mitochondrial impairment. We then concentrate on redox-active agents, with mild-redox potential, who shuttle electrons among specific cytosolic or mitochondrial redox-centers. We propose that specific redox agents with mild redox potential (-0.1 V; 0.1 V) improve mitochondrial function because they can readily donate or accept electrons in biological systems, thus they enhance metabolic activity and prevent reactive oxygen species (ROS) production. These agents are likely to lack toxic effects because they lack the risk of inhibiting electron transfer in redox centers. This is different from redox agents with strong negative (-0.4 V; -0.2 V) or positive (0.2 V; 0.4 V) redox potentials who alter the redox status of redox-centers (i.e., become permanently reduced or oxidized). This view has been demonstrated by testing the effect of several redox active agents on cellular senescence. Methylene blue (MB, redox potential ≅10 mV) appears to readily cycle between the oxidized and reduced forms using specific mitochondrial and cytosolic redox centers. MB is most effective in delaying cell senescence and enhancing mitochondrial function in vivo and in vitro. Mild-redox agents can alter the biochemical activity of specific mitochondrial components, which then in response alters the expression of nuclear and mitochondrial genes. We present the concept of mitochondrial electron-carrier bypass as a potential result of mild-redox agents, a method to prevent ROS production, improve mitochondrial function, and delay cellular aging. Thus, mild-redox agents may prevent/delay mitochondria-driven disorders. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

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

    PubMed Central

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

    2017-01-01

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

  13. 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

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

    PubMed

    Radius, R L

    1981-07-01

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

  15. Insights into the post-transcriptional regulation of the mitochondrial electron transport chain.

    PubMed

    Sirey, Tamara M; Ponting, Chris P

    2016-10-15

    The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. © 2016 The Author(s).

  16. Insights into the post-transcriptional regulation of the mitochondrial electron transport chain

    PubMed Central

    Sirey, Tamara M.; Ponting, Chris P.

    2016-01-01

    The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. PMID:27911731

  17. The rescue of microtubule-dependent traffic recovers mitochondrial function in Parkinson's disease.

    PubMed

    Esteves, A R; Gozes, I; Cardoso, S M

    2014-01-01

    In Parkinson's disease mitochondrial dysfunction can lead to a deficient ATP supply to microtubule protein motors leading to mitochondrial axonal transport disruption. Compromised axonal transport will then lead to a disorganized distribution of mitochondria and other organelles in the cell, as well as, the accumulation of aggregated proteins like alpha-synuclein. Moreover, axonal transport disruption can trigger synaptic accumulation of autophagosomes packed with damaged mitochondria and protein aggregates promoting synaptic failure. We previously observed that neuronal-like cells with an inherent mitochondrial impairment derived from PD patients contain a disorganized microtubule network, as well as, alpha-synuclein oligomer accumulation. In this work we provide new evidence that an agent that promotes microtubule network assembly, NAP (davunetide), improves microtubule-dependent traffic, restores the autophagic flux and potentiates autophagosome-lysosome fusion leading to autophagic vacuole clearance in Parkinson's disease cells. Moreover, NAP is capable of efficiently reducing alpha-synuclein oligomer content and its sequestration by the mitochondria. Most interestingly, NAP decreases mitochondrial ubiquitination levels, as well as, increases mitochondrial membrane potential indicating a rescue in mitochondrial function. Overall, we demonstrate that by improving microtubule-mediated traffic, we can avoid mitochondrial-induced damage and thus recover cell homeostasis. These results prove that NAP may be a promising therapeutic lead candidate for neurodegenerative diseases that involve axonal transport failure and mitochondrial impairment as hallmarks, like Parkinson's disease and related disorders.

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

    PubMed Central

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

    1998-01-01

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

  19. Liver mitochondrial dysfunction and electron transport chain defect induced by high dietary copper in broilers.

    PubMed

    Yang, Fan; Cao, Huabin; Su, Rongsheng; Guo, Jianying; Li, Chengmei; Pan, Jiaqiang; Tang, Zhaoxin

    2017-09-01

    Copper is an important trace mineral in the diet of poultry due to its biological activity. However, limited information is available concerning the effects of high copper on mitochondrial dysfunction. In this study, 72 broilers were used to investigate the effects of high dietary copper on liver mitochondrial dysfunction and electron transport chain defect. Birds were fed with different concentrations [11, 110, 220, and 330 mg of copper/kg dry matter (DM)] of copper from tribasic copper chloride (TBCC). The experiment lasted for 60 d. Liver tissues on d 60 were subjected to histopathological observation. Additionally, liver mitochondrial function was recorded on d 12, 36, and 60. Moreover, a site-specific defect in the electron transport chain in liver mitochondria was also identified by using various chemical inhibitors of mitochondrial respiration. The results showed different degrees of degeneration, mitochondrial swelling, and high-density electrons in hepatocytes. In addition, the respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) in liver mitochondria increased at first and then decreased in high-dose groups. Moreover, hydrogen peroxide (H2O2) generation velocity in treated groups was higher than that in control group, which were magnified by inhibiting electron transport at Complex IV. The results indicated that high dietary copper could decline liver mitochondrial function in broilers. The presence of a site-specific defect at Complex IV in liver mitochondria may be responsible for liver mitochondrial dysfunction caused by high dietary copper. © 2017 Poultry Science Association Inc.

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Shaw, Jillian L.; Chang, Karen T.

    2013-01-01

    Post-mortem brains from Down syndrome (DS) and Alzheimer's disease (AD) patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1), but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, we explored the functional interaction between DSCR1 and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. We find that the Drosophila homolog of DSCR1, Nebula, delays neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, we show that Nebula upregulation protects against axonal transport defects by restoring calcineurin and GSK-3β signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, our findings imply that correcting calcineurin and GSK-3β signaling can prevent APP-induced pathologies. Our data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD. PMID:24086147

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

    PubMed

    Shaw, Jillian L; Chang, Karen T

    2013-01-01

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

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

    PubMed

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

    1993-09-16

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

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

    PubMed

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

    1990-02-01

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

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

    PubMed

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

    2017-03-01

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

  7. 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.

  8. Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

    PubMed

    Trotta, Andrew P; Gelles, Jesse D; Serasinghe, Madhavika N; Loi, Patrick; Arbiser, Jack L; Chipuk, Jerry E

    2017-07-14

    The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAF(V600E)), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAF(V600E) is pharmacologically inhibited by targeted therapies (e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAF(V600E) melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

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

    PubMed

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

    1993-09-01

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

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

    NASA Astrophysics Data System (ADS)

    Huang, Feimin; Li, Xing; Zhang, Yinglong

    2017-07-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2017-04-15

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

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

    PubMed

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

    2017-08-08

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

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

    PubMed

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

    2017-06-01

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

  15. Lipophilic Triphenylphosphonium Cations Inhibit Mitochondrial Electron Transport Chain and Induce Mitochondrial Proton Leak

    PubMed Central

    Anděl, Michal

    2015-01-01

    Background The lipophilic positively charged moiety of triphenylphosphonium (TPP+) has been used to target a range of biologically active compounds including antioxidants, spin-traps and other probes into mitochondria. The moiety itself, while often considered biologically inert, appears to influence mitochondrial metabolism. Methodology/Principal Findings We used the Seahorse XF flux analyzer to measure the effect of a range of alkylTPP+ on cellular respiration and further analyzed their effect on mitochondrial membrane potential and the activity of respiratory complexes. We found that the ability of alkylTPP+ to inhibit the respiratory chain and decrease the mitochondrial membrane potential increases with the length of the alkyl chain suggesting that hydrophobicity is an important determinant of toxicity. Conclusions/Significance More hydrophobic TPP+ derivatives can be expected to have a negative impact on mitochondrial membrane potential and respiratory chain activity in addition to the effect of the biologically active moiety attached to them. Using shorter linker chains or adding hydrophilic functional groups may provide a means to decrease this negative effect. PMID:25927600

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

    PubMed

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

    2017-01-01

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

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

    PubMed

    Kuznetsov, A V

    2013-01-01

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

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

    SciTech Connect

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

    1984-03-01

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

  19. 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.

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

    PubMed Central

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

    2016-01-01

    Recent studies have highlighted the fact that cancer cells have an altered metabolic phenotype, and this metabolic reprogramming is required to drive biosynthesis pathways necessary for rapid replication and proliferation. Specifically, the importance of tricarboxylic acid (TCA) cycle-generated intermediates in the regulation of cancer cells proliferation has been recently appreciated. One function of monocarboxylate transporters (MCTs) is to transport the TCA 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. Here, 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 to lactate. Pyruvate supplementation fueled 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-fueled mitochondrial respiration. PMID:22458763

  1. 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.

  2. Insights into the Mutation-Induced HHH Syndrome from Modeling Human Mitochondrial Ornithine Transporter-1

    PubMed Central

    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

  3. 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

  4. Measurement of mitochondrial Ca2+ transport mediated by three transport proteins: VDAC1, the Na+/Ca2+ exchanger, and the Ca2+ uniporter.

    PubMed

    Ben-Hail, Danya; Palty, Raz; Shoshan-Barmatz, Varda

    2014-02-01

    Ca(2+) is a ubiquitous cellular signal, with changes in intracellular Ca(2+) concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca(2+) uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca(2+) signaling, energy metabolism and cell death. Ca(2+) transport across the inner and outer mitochondrial membranes is mediated by several proteins, including channels, antiporters, and a uniporter. In this article, we present the background to several methods now established for assaying mitochondrial Ca(2+) transport activity across both mitochondrial membranes. The first of these is Ca(2+) transport mediated by the outer mitochondrial protein, the voltage-dependent anion-selective channel protein 1 (VDAC1, also known as porin 1), both as a purified protein reconstituted into a planar lipid bilayer (PLB) or into liposomes and as a mitochondrial membrane-embedded protein. The second method involves isolated mitochondria for assaying the activity of an inner mitochondrial membrane transport protein, the mitochondrial Ca(2+) uniporter (MCU) that transports Ca(2+) and is powered by the steep mitochondrial membrane potential. In the event of Ca(2+) overload, this leads to opening of the mitochondrial permeability transition pore (MPTP) and cell death. The third method describes how Na(+)-dependent mitochondrial Ca(2+) efflux mediated by mitochondrial NCLX, a member of the Na(+)/Ca(2+) exchanger superfamily, can be assayed in digitonin-permeabilized HEK-293 cells. The Ca(2+)-transport assays can be performed under various conditions and in combination with inhibitors, allowing detailed characterization of the transport activity of interest.

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

    PubMed Central

    LePard, Kathy J; Cellini, Joseph

    2013-01-01

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

  6. Biguanides sensitize leukemia cells to ABT-737-induced apoptosis by inhibiting mitochondrial electron transport

    PubMed Central

    Velez, Juliana; Pan, Rongqing; Lee, Jason T.C.; Enciso, Leonardo; Suarez, Marta; Duque, Jorge Eduardo; Jaramillo, Daniel; Lopez, Catalina; Morales, Ludis; Bornmann, William; Konopleva, Marina; Krystal, Gerald; Andreeff, Michael; Samudio, Ismael

    2016-01-01

    Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax. PMID:27283492

  7. Biguanides sensitize leukemia cells to ABT-737-induced apoptosis by inhibiting mitochondrial electron transport.

    PubMed

    Velez, Juliana; Pan, Rongqing; Lee, Jason T C; Enciso, Leonardo; Suarez, Marta; Duque, Jorge Eduardo; Jaramillo, Daniel; Lopez, Catalina; Morales, Ludis; Bornmann, William; Konopleva, Marina; Krystal, Gerald; Andreeff, Michael; Samudio, Ismael

    2016-08-09

    Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax.

  8. IFPA meeting 2015 workshop report I: placental mitochondrial function, transport systems and epigenetics.

    PubMed

    Bianco-Miotto, T; Blundell, C; Buckberry, S; Chamley, L; Chong, S; Cottrell, E; Dawson, P; Hanna, C; Holland, O; Lewis, R M; Moritz, K; Myatt, L; Perkins, A V; Powell, T; Saffery, R; Sferruzzi-Perri, A; Sibley, C; Simmons, D; O'Tierney-Ginn, P F

    2016-12-01

    Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops covered areas of placental regulation and nutrient handling: 1) placental epigenetics; 2) placental mitochondrial function; 3) placental transport systems.

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

    PubMed Central

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

    2013-01-01

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

  10. Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein.

    PubMed

    Giangregorio, Nicola; Tonazzi, Annamaria; Console, Lara; Indiveri, Cesare

    2017-02-01

    The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD(+). After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.

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

    PubMed

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

    2011-07-01

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

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

    PubMed Central

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

    2011-01-01

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

  13. 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.

  14. The axonal cytoskeleton: from organization to function

    PubMed Central

    Kevenaar, Josta T.; Hoogenraad, Casper C.

    2015-01-01

    The axon is the single long fiber that extends from the neuron and transmits electrical signals away from the cell body. The neuronal cytoskeleton, composed of microtubules (MTs), actin filaments and neurofilaments, is not only required for axon formation and axonal transport but also provides the structural basis for several specialized axonal structures, such as the axon initial segment (AIS) and presynaptic boutons. Emerging evidence suggest that the unique cytoskeleton organization in the axon is essential for its structure and integrity. In addition, the increasing number of neurodevelopmental and neurodegenerative diseases linked to defect in actin- and microtubule-dependent processes emphasizes the importance of a properly regulated cytoskeleton for normal axonal functioning. Here, we provide an overview of the current understanding of actin and microtubule organization within the axon and discuss models for the functional role of the cytoskeleton at specialized axonal structures. PMID:26321907

  15. The axonal cytoskeleton: from organization to function.

    PubMed

    Kevenaar, Josta T; Hoogenraad, Casper C

    2015-01-01

    The axon is the single long fiber that extends from the neuron and transmits electrical signals away from the cell body. The neuronal cytoskeleton, composed of microtubules (MTs), actin filaments and neurofilaments, is not only required for axon formation and axonal transport but also provides the structural basis for several specialized axonal structures, such as the axon initial segment (AIS) and presynaptic boutons. Emerging evidence suggest that the unique cytoskeleton organization in the axon is essential for its structure and integrity. In addition, the increasing number of neurodevelopmental and neurodegenerative diseases linked to defect in actin- and microtubule-dependent processes emphasizes the importance of a properly regulated cytoskeleton for normal axonal functioning. Here, we provide an overview of the current understanding of actin and microtubule organization within the axon and discuss models for the functional role of the cytoskeleton at specialized axonal structures.

  16. Cobalt inhibits motility of axonal mitochondria and induces axonal degeneration in cultured dorsal root ganglion cells of rat.

    PubMed

    Kikuchi, Shin; Ninomiya, Takafumi; Kohno, Takayuki; Kojima, Takashi; Tatsumi, Haruyuki

    2017-06-27

    Cobalt is a trace element that localizes in the human body as cobalamin, also known as vitamin B12. Excessive cobalt exposure induces a peripheral neuropathy, the mechanisms of which are yet to be elucidated. We investigated how cobalt may affect mitochondrial motility in primary cultures of rat dorsal root ganglion (DRG). We observed mitochondrial motility by time-lapse imaging after DsRed2 tagging via lentivirus, mitochondrial structure using transmission electron microscopy (TEM), and axonal swelling using immunocytochemical staining. The concentration of cobaltous ion (Co(2+)) required to significantly suppress mitochondrial motility is lower than that required to induce axonal swelling following a 24-h treatment. Exposure to relatively low concentrations of Co(2+) for 48 h suppressed mitochondrial motility without leading to axonal swelling. TEM images indicated that Co(2+) induces mitochondrial destruction. Our results show that destruction of the axonal mitochondria precedes the axonal degeneration induced by Co(2+) exposure.

  17. Paradoxical effect of mitochondrial respiratory chain impairment on insulin signaling and glucose transport in adipose cells.

    PubMed

    Shi, Xiarong; Burkart, Alison; Nicoloro, Sarah M; Czech, Michael P; Straubhaar, Juerg; Corvera, Silvia

    2008-11-07

    Adipocyte function is crucial for the control of whole body energy homeostasis. Pathway analysis of differentiating 3T3-L1 adipocytes reveals that major metabolic pathways induced during differentiation involve mitochondrial function. However, it is not clear why differentiated white adipocytes require enhanced respiratory chain activity relative to pre-adipocytes. To address this question, we used small interference RNA to interfere with the induction of the transcription factor Tfam, which is highly induced between days 2 and 4 of differentiation and is crucial for replication of mitochondrial DNA. Interference with Tfam resulted in cells with decreased respiratory chain capacity, reflected by decreased basal oxygen consumption, and decreased mitochondrial ATP synthesis, but no difference in many other adipocyte functions or expression levels of adipose-specific genes. However, insulin-stimulated GLUT4 translocation to the cell surface and subsequent glucose transport are impaired in Tfam knockdown cells. Paradoxically, insulin-stimulated Akt phosphorylation is significantly enhanced in these cells. These studies reveal independent links between mitochondrial function, insulin signaling, and glucose transport, in which impaired respiratory chain activity enhances insulin signaling to Akt phosphorylation, but impairs GLUT4 translocation. These results indicate that mitochondrial respiratory chain dysfunction in adipocytes can cause impaired insulin responsiveness of GLUT4 translocation by a mechanism downstream of the Akt protein kinase.

  18. Inner membrane fusion mediates spatial distribution of axonal mitochondria

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  19. Inner membrane fusion mediates spatial distribution of axonal mitochondria.

    PubMed

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

    2016-01-08

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

  20. Inner membrane fusion mediates spatial distribution of axonal mitochondria

    PubMed Central

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

    2016-01-01

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

  1. Effect of thyroid hormones and their analogues on the mitochondrial calcium transport activity.

    PubMed

    De Giovanni, R; Asta, L; Covello, C; Marotta, M; Mazzulla, S; Parrilla, R; Pitrelli, G; Spena, A; Martino, G

    1992-01-01

    In this paper the authors studied the effects of thyroid hormones and their structural analogues on the mitochondrial calcium transport activities. The thyroid hormones, 3,5,3' L-triiodothyronine (LT3) and 3,5,3'5' L-tetraiodothyronine (LT4) at physiological intracellular concentrations between 7.2 and 9 nM, decouple total Ca++ transport, as well as inhibit the passive transport of Ca++, either due to oxidation of pyruvate, malate or succinate or after inhibition with rotenone. The optical isomers 3,5,3' D-triiodothyronine (DT3) and 3,5,3',5' D-tetraiodothyronine (DT4) are less effective at all the used concentrations. Furthermore the structural analogues 3,3',5' L-triiodothyronine (LrT3), 3,5-dicloro, 3',5' L-diiodothyronine (LDiClT2) and 3,5 L-diiodothyronine (LT2) furnished even less effects on the same activities. The effect of the thyroid hormones and of their structural analogues has revealed that the mitochondrial calcium transport may be influenced both by a stereospecific interaction between hormones and protein ligands and by a lipophilic chaotropic action on the mitochondrial membranes lipids. In this context it is interesting to consider that both thyroid hormones and Ca++ transport activity are interacting with the energetic metabolism by means of phosphorylation and substrate oxidation mechanism.

  2. Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate.

    PubMed

    Marobbio, C M T; Vozza, A; Harding, M; Bisaccia, F; Palmieri, F; Walker, J E

    2002-11-01

    The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.

  3. Is multiple sclerosis a mitochondrial disease?

    PubMed Central

    Mao, Peizhong; Reddy, P. Hemachandra

    2009-01-01

    Multiple sclerosis (MS) is a relatively common and etiologically unknown disease with no cure treatment. It is the leading cause of neurological disability in young adults, affecting over two million people worldwide. Traditionally, MS has been considered a chronic, inflammatory disorder of the central white matter in which ensuing demyelination results in physical disability. Recently, MS has become increasingly viewed as a neurodegenerative disorder in which axonal injury, neuronal loss, and atrophy of the central nervous system lead to permanent neurological and clinical disability. In this article, we discuss the latest developments on MS research, including etiology, pathology, genetic association, EAE animal models, mechanisms of neuronal injury and axonal transport and therapeutics. In this article, we also focus on the mechanisms of mitochondrial dysfunction that are involved in MS, including mitochondrial DNA defects, and mitochondrial structural/functional changes. PMID:19607913

  4. Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration

    PubMed Central

    Zhang, Jian-Nan; Michel, Uwe; Lenz, Christof; Friedel, Caroline C.; Köster, Sarah; d’Hedouville, Zara; Tönges, Lars; Urlaub, Henning; Bähr, Mathias; Lingor, Paul; Koch, Jan C.

    2016-01-01

    Axonal degeneration is a key initiating event in many neurological diseases. Focal lesions to axons result in a rapid disintegration of the perilesional axon by acute axonal degeneration (AAD) within several hours. However, the underlying molecular mechanisms of AAD are only incompletely understood. Here, we studied AAD in vivo through live-imaging of the rat optic nerve and in vitro in primary rat cortical neurons in microfluidic chambers. We found that calpain is activated early during AAD of the optic nerve and that calpain inhibition completely inhibits axonal fragmentation on the proximal side of the crush while it attenuates AAD on the distal side. A screening of calpain targets revealed that collapsin response mediator protein-2 (CRMP2) is a main downstream target of calpain activation in AAD. CRMP2-overexpression delayed bulb formation and rescued impairment of axonal mitochondrial transport after axotomy in vitro. In vivo, CRMP2-overexpression effectively protected the proximal axon from fragmentation within 6 hours after crush. Finally, a proteomic analysis of the optic nerve was performed at 6 hours after crush, which identified further proteins regulated during AAD, including several interactors of CRMP2. These findings reveal CRMP2 as an important mediator of AAD and define it as a putative therapeutic target. PMID:27845394

  5. Hydrogen sulfide-mediated stimulation of mitochondrial electron transport involves inhibition of the mitochondrial phosphodiesterase 2A, elevation of cAMP and activation of protein kinase A.

    PubMed

    Módis, Katalin; Panopoulos, Panagiotis; Coletta, Ciro; Papapetropoulos, Andreas; Szabo, Csaba

    2013-11-01

    Although hydrogen sulfide (H₂S) is generally known as a mitochondrial poison, recent studies show that lower concentrations of H₂S play a physiological role in the stimulation of mitochondrial electron transport and cellular bioenergetics. This effect involves electron donation at Complex II. Other lines of recent studies demonstrated that one of the biological actions of H₂S involves inhibition of cAMP and cGMP phosphodiesterases (PDEs). Given the emerging functional role of the mitochondrial isoform of cAMP PDE (PDE2A) in the regulation of mitochondrial function the current study investigated whether cAMP-dependent mechanisms participate in the stimulatory effect of NaHS on mitochondrial function. In isolated rat liver mitochondria, partial digestion studies localized PDE2A into the mitochondrial matrix. NaHS exerted a concentration-dependent inhibitory effect on recombinant PDE2A enzyme in vitro. Moreover, NaHS induced an elevation of cAMP levels when added to isolated mitochondria and stimulated the mitochondrial electron transport. The latter effect was inhibited by Rp-cAMP, an inhibitor of the cAMP-dependent protein kinase (PKA). The current findings suggest that the direct electron donating effect of NaHS is amplified by an intramitochondrial cAMP system, which may involve the inhibition of PDE2A and subsequent, cAMP-mediated stimulation of PKA.

  6. 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

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

    PubMed Central

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

    2015-01-01

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

  8. 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

  9. Glutamate transport decreases mitochondrial pH and modulates oxidative metabolism in astrocytes.

    PubMed

    Azarias, Guillaume; Perreten, Hélène; Lengacher, Sylvain; Poburko, Damon; Demaurex, Nicolas; Magistretti, Pierre J; Chatton, Jean-Yves

    2011-03-09

    During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca(2+)-mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

  10. 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.

  11. Model-based confirmation of alternative substrates of mitochondrial electron transport chain.

    PubMed

    Kleessen, Sabrina; Araújo, Wagner L; Fernie, Alisdair R; Nikoloski, Zoran

    2012-03-30

    Discrimination of metabolic models based on high throughput metabolomics data, reflecting various internal and external perturbations, is essential for identifying the components that contribute to the emerging behavior of metabolic processes. Here, we investigate 12 different models of the mitochondrial electron transport chain (ETC) in Arabidopsis thaliana during dark-induced senescence in order to elucidate the alternative substrates to this metabolic pathway. Our findings demonstrate that the coupling of the proposed computational approach, based on dynamic flux balance analysis, with time-resolved metabolomics data results in model-based confirmations of the hypotheses that, during dark-induced senescence in Arabidopsis, (i) under conditions where the main substrate for the ETC are not fully available, isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase are able to donate electrons to the ETC, (ii) phytanoyl-CoA does not act even as an indirect substrate of the electron transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex, and (iii) the mitochondrial γ-aminobutyric acid transporter has functional significance in maintaining mitochondrial metabolism. Our study provides a basic framework for future in silico studies of alternative pathways in mitochondrial metabolism under extended darkness whereby the role of its components can be computationally discriminated based on available molecular profile data.

  12. Model-based Confirmation of Alternative Substrates of Mitochondrial Electron Transport Chain

    PubMed Central

    Kleessen, Sabrina; Araújo, Wagner L.; Fernie, Alisdair R.; Nikoloski, Zoran

    2012-01-01

    Discrimination of metabolic models based on high throughput metabolomics data, reflecting various internal and external perturbations, is essential for identifying the components that contribute to the emerging behavior of metabolic processes. Here, we investigate 12 different models of the mitochondrial electron transport chain (ETC) in Arabidopsis thaliana during dark-induced senescence in order to elucidate the alternative substrates to this metabolic pathway. Our findings demonstrate that the coupling of the proposed computational approach, based on dynamic flux balance analysis, with time-resolved metabolomics data results in model-based confirmations of the hypotheses that, during dark-induced senescence in Arabidopsis, (i) under conditions where the main substrate for the ETC are not fully available, isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase are able to donate electrons to the ETC, (ii) phytanoyl-CoA does not act even as an indirect substrate of the electron transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex, and (iii) the mitochondrial γ-aminobutyric acid transporter has functional significance in maintaining mitochondrial metabolism. Our study provides a basic framework for future in silico studies of alternative pathways in mitochondrial metabolism under extended darkness whereby the role of its components can be computationally discriminated based on available molecular profile data. PMID:22334689

  13. 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.

  14. Mitochondria Localize to Injured Axons to Support Regeneration.

    PubMed

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

    2016-12-21

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

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

    PubMed Central

    Zhang, Jingyi; McDonald, Alexander J.

    2016-01-01

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

  16. Amiloride and diltiazem inhibition of microsomal and mitochondrial Na+ and Ca2+ transport.

    PubMed

    Sordahl, L A; LaBelle, E F; Rex, K A

    1984-01-01

    Amiloride, a K+-sparing diuretic, and diltiazem, a Ca2+ channel antagonist, have both been reported to inhibit Na+ transport-associated processes in different subcellular membrane systems. In this report, similar inhibitory effects of both agents are demonstrated on Na+-induced Ca2+ release from rabbit heart mitochondria and on Na+ uptake in a kidney medulla microsomal preparation. Both amiloride and diltiazem produce 50% inhibition of Na+ uptake in kidney microsomes at the same concentrations. Heart mitochondrial Na+-induced Ca2+ release was 50% inhibited by 6 microM diltiazem and 200 microM amiloride. No effects of either agent on mitochondrial respiratory activity were observed. The results suggest a specific effect of both drugs on a Na+-binding site associated with an antiport exchange process. These data also extend previous observations suggesting the use of these agents as tools to define further ion transport mechanisms in biological membranes.

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

    PubMed

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

    2016-05-01

    The mitochondrial carrier protein MttA is involved in the biosynthesis of itaconic acid in Aspergillus terreus. In this paper, the transport specificity of MttA is analyzed making use of different metabolically engineered Aspergillus niger strains. Furthermore, the mitochondrial localization of this protein is confirmed using fluorescence microscopy. It was found that MttA preferentially transports cis-aconitic acid over citric acid and does not transport itaconic acid. The expression of MttA in selected A. niger strains results in secretion of aconitic acid. MttA can be used in further strain engineering strategies to transport cis-aconitic acid to the cytosol to produce itaconic acid or related metabolites. The microbial production of aconitic acid (9g/L) is achieved in strains expressing this transport protein. Thus, metabolic engineering can be used for both the in vivo characterization of transport protein function like MttA and to make use of this protein by creating aconitic acid producing strains. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  18. Fourier imaging correlation spectroscopy: Technique development and application to colloidal thin films and intracellular mitochondrial transport

    NASA Astrophysics Data System (ADS)

    Knowles, Michelle Kay

    2003-10-01

    Understanding fluid dynamics is fundamentally intriguing and relevant to many areas of applied science, including polymer materials and cellular transport. Many complex fluids are difficult to study using traditional methods, which are limited in sensitivity, dynamic range or spatial information. In this work, a new technique, Fourier Imaging Correlation Spectroscopy (FICS), is developed in order to measure the dynamics of complex fluids over a broad dynamic range with high sensitivity. FICS measures complex fluid structure one length scale at a time and allows for direct calculation of the intermediate scattering function; a function that describes how the system is changing on a given length scale as a function of time. The sensitivity of FICS allows for study of materials with intrinsically low signals, such as thin films. Colloidal thin film measurements provided a proof-of-principle of FICS by comparing the intermediate scattering function calculated from FICS data to results from an established technique, digital video microscopy. FICS is an ideal method for obtaining information about mitochondrial transport within living cells. Mitochondrial dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins. This leads to complex multi-exponential relaxations occurring over a wide range of spatial and temporal scales. The cytoskeleton consists of an interconnected polymer network whose primary components are microfilaments and microtubules. Cytoskeletal filaments work with motor proteins to traffic organelles within the cell. Components of the cytoskeleton were selectively destabilized and the resulting mitochondrial dynamics measured using FICS and digital video microscopy. These studies show that both microfilaments and microtubules are necessary for transport of the mitochondrial reticulum. FICS measurements reveal that microfilaments control short-range (0.8--1.6 mum) dynamics and microtubules are

  19. Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

    PubMed Central

    Sanders, S P; Zweier, J L; Kuppusamy, P; Harrison, S J; Bassett, D J; Gabrielson, E W; Sylvester, J T

    1993-01-01

    Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death. PMID:8380815

  20. 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.

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

    PubMed

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

    2015-09-01

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

  2. Why do axons differ in caliber?

    PubMed

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

    2012-01-11

    CNS axons differ in diameter (d) by nearly 100-fold (∼0.1-10 μm); therefore, they differ in cross-sectional area (d(2)) 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 d(2). We asked, given constraints on space and energy, 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 the following: (1) thin axons are most numerous; (2) mean firing frequencies, estimated for nine of the identified axon classes, are low for thin fibers and high for thick ones, ranging from ∼1 to >100 Hz; (3) 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; and (4) mitochondrial volume/axon length rises ≥d(2). To explain the pressure toward 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.

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

    PubMed Central

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

    2012-01-01

    Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP–like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality. PMID:23209432

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

    PubMed Central

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

    2014-01-01

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

  5. Mechanisms for the transport of alpha,omega-dicarboxylates through the mitochondrial inner membrane.

    PubMed

    Liu, G; Hinch, B; Beavis, A D

    1996-10-11

    alpha,omega-Dicarboxylates have antibacterial properties, have been used in the treatment of hyperpigmentary disorders, are active against various melanoma cell lines, and can also undergo beta-oxidation. Little, however, is known about their transport. In this paper, we examine the mitochondrial transport of alpha, omega-dicarboxylates ranging from oxalate (DC2) to sebacate (DC10). DC2-DC10 are transported by the inner membrane anion channel (IMAC). DC6-DC10 are also transported by an electroneutral mechanism that appears to reflect transport of the acid through the lipid bilayer. At 37 degrees C and pH 7.0, DC10 is transported very rapidly at 3 micromol/min.mg, and respiring mitochondria swell in the K+ salts of these acids. This transport mechanism is probably the major pathway by which the longer dicarboxylates enter cells, bacteria, and mitochondria. We also demonstrate that DC5-DC10 can also be transported by an electroneutral mechanism mediated by tributyltin, a potent inhibitor of IMAC. The mechanism appears to involve electroneutral exchange of a TBT-dicarboxylate-H complex for TBT-OH. Finally, we present evidence that of all the dicarboxylates tested only DC2-DC4 can be transported by the classical dicarboxylate carrier.

  6. Gem1 and ERMES do not directly affect phosphatidylserine transport from ER to mitochondria or mitochondrial inheritance.

    PubMed

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

    2012-06-01

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

  7. Rotenone-induced Impairment of Mitochondrial Electron Transport Chain Confers a Selective Priming Signal for NLRP3 Inflammasome Activation*

    PubMed Central

    Won, Ji-Hee; Park, Sangjun; Hong, Sujeong; Son, Seunghwan; Yu, Je-Wook

    2015-01-01

    Mitochondrial dysfunction is considered crucial for NLRP3 inflammasome activation partly through its release of mitochondrial toxic products, such as mitochondrial reactive oxygen species (mROS)2 and mitochondrial DNA (mtDNA). Although previous studies have shown that classical NLRP3-activating stimulations lead to mROS generation and mtDNA release, it remains poorly understood whether and how mitochondrial damage-derived factors may contribute to NLRP3 inflammasome activation. Here, we demonstrate that impairment of the mitochondrial electron transport chain by rotenone primes NLRP3 inflammasome activation only upon costimulation with ATP and not with nigericin or alum. Rotenone-induced priming of NLRP3 in the presence of ATP triggered the formation of specklike NLRP3 or ASC aggregates and the association of NLRP3 with ASC, resulting in NLRP3-dependent caspase-1 activation. Mechanistically, rotenone confers a priming signal for NLRP3 inflammasome activation only in the context of aberrant high-grade, but not low-grade, mROS production and mitochondrial hyperpolarization. By contrast, rotenone/ATP-mediated mtDNA release and mitochondrial depolarization are likely to be merely an indication of mitochondrial damage rather than triggering factors for NLRP3 inflammasome activation. Our results provide a molecular insight into the selective contribution made by mitochondrial dysfunction to the NLRP3 inflammasome pathway. PMID:26416893

  8. Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

    PubMed Central

    2011-01-01

    To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity. Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a

  9. Selenium and zinc protect brain mitochondrial antioxidants and electron transport chain enzymes following postnatal protein malnutrition.

    PubMed

    Adebayo, Olusegun L; Adenuga, Gbenga A; Sandhir, Rajat

    2016-05-01

    Selenium (Se) and zinc (Zn) are trace elements required for optimal brain functions. Thus, the role of Se and Zn against protein malnutrition induced oxidative stress on mitochondrial antioxidants and electron transport chain (ETC) enzymes from rats' brain were investigated. Normal protein (NP) and low protein (LP) rats were fed with diets containing 16% and 5% casein respectively for a period of 10weeks. Then the rats were supplemented with Se and Zn at a concentration of 0.15mgL(-1) and 227mgL(-1) in drinking water for 3weeks after which the rats were sacrificed. The results obtained from the study showed significant (p<0.05) increase in lipid peroxidation (LPO), ROS production, oxidized glutathione (GSSG) levels and mitochondrial swelling and significant (p<0.05) reductions in catalase (CAT) and Mn-superoxide dismutase (Mn-SOD) activities, glutathione (GSH) levels, GSH/GSSG ratio and MTT reduction as a result of LP ingestion. The activities of mitochondrial ETC enzymes were also significantly inhibited in both the cortex and cerebellum of LP-fed rats. Supplementation with either Se or Zn restored the alterations in all the parameters. The study showed that Se and Zn might be beneficial in protecting mitochondrial antioxidants and ETC enzymes against protein malnutrition induced oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-12-01

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

  11. Three conserved histidine residues contribute to mitochondrial iron transport through mitoferrins.

    PubMed

    Brazzolotto, Xavier; Pierrel, Fabien; Pelosi, Ludovic

    2014-05-15

    Iron is an essential element for almost all organisms. In eukaryotes, it is mainly used in mitochondria for the biosynthesis of iron-sulfur clusters and haem group maturation. Iron is delivered into the mitochondrion by mitoferrins, members of the MCF (mitochondrial carrier family), through an unknown mechanism. In the present study, the yeast homologues of these proteins, Mrs3p (mitochondrial RNA splicing 3) and Mrs4p, were studied by inserting them into liposomes. In this context, they could transport Fe2+ across the proteoliposome membrane, as shown using the iron chelator bathophenanthroline. A series of amino acid-modifying reagents were screened for their effects on Mrs3p-mediated iron transport. The results of the present study suggest that carboxy and imidazole groups are essential for iron transport. This was confirmed by in vivo complementation assays, which demonstrated that three highly conserved histidine residues are important for Mrs3p function. These histidine residues are not conserved in other MCF members and thus they are likely to play a specific role in iron transport. A model describing how these residues help iron to transit smoothly across the carrier cavity is proposed and compared with the structural and biochemical data available for other carriers in this family.

  12. Pioglitazone halts axonal degeneration in a mouse model of X-linked adrenoleukodystrophy.

    PubMed

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

    2013-08-01

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

  13. Pioglitazone halts axonal degeneration in a mouse model of X-linked adrenoleukodystrophy

    PubMed Central

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

    2013-01-01

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

  14. 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.

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

    PubMed Central

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

    2015-01-01

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

  16. 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

  17. 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.

  18. 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

  19. The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids*

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-05-09

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

  1. 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

  2. Increased mitochondrial ROS formation by acetaminophen in human hepatic cells is associated with gene expression changes suggesting disruption of the mitochondrial electron transport chain.

    PubMed

    Jiang, Jian; Briedé, Jacob J; Jennen, Danyel G J; Van Summeren, Anke; Saritas-Brauers, Karen; Schaart, Gert; Kleinjans, Jos C S; de Kok, Theo M C M

    2015-04-16

    Acetaminophen (APAP) overdosage results in hepatotoxicity, but the underlying molecular mechanisms are still not completely understood. In the current study, we focused on mitochondrial-specific oxidative liver injury induced by APAP exposure. Owning to genetic polymorphisms in the CYP2E1 gene or varying inducibility by xenobiotics, the CYP2E1 mRNA level and protein activity vary extensively among individuals. As CYP2E1 is a known ROS generating enzyme, we chose HepG2 to minimize CYP2E1-induced ROS formation, which will help us better understand the APAP induced mitochondrial-specific hepatotoxicity in a subpopulation with low CYP2E1 activity. HepG2 cells were exposed to a low and toxic dose (0.5 and 10mM) of APAP and analyzed at four time points for genome-wide gene expression. Mitochondria were isolated and electron spin resonance spectroscopy was performed to measure the formation of mitochondrial ROS. The yield of ATP was measured to confirm the impact of the toxic dose of APAP on cellular energy production. Our results indicate that 10mM APAP significantly influences the expression of mitochondrial protein-encoding genes in association with an increase in mitochondrial ROS formation. Additionally, 10mM APAP affects the expression of genes encoding the subunits of electron transport chain (ETC) complexes, which may alter normal mitochondrial functions by disrupting the assembly, stability, and structural integrity of ETC complexes, leading to a measurable depletion of ATP, and cell death. The expression of mitochondrium-specific antioxidant enzyme, SOD2, is reduced which may limit the ROS scavenging ability and cause imbalance of the mitochondrial ROS homeostasis. Overall, transcriptome analysis reveals the molecular processes involved in the observed APAP-induced increase of mitochondrial ROS formation and the associated APAP-induced oxidative stress.

  3. Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state.

    PubMed

    Moini, H; Arroyo, A; Vaya, J; Packer, L

    1999-01-01

    The polyphenolic structure common to flavonoids enables them to donate electrons and exert antioxidant activity. Since the mitochondrial electron transport chain consists of a series of redox intermediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated on the rat liver mitochondrial electron transport chain. A French maritime pine bark extract (PBE), a complex mixture of polyphenols and related pure flavonoids, was able to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group; the donated electrons can be utilized by cytochrome c oxidase. Among single flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitively inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent Km value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-)-epicatechin gallate showed minor inhibitory effects, while (+/-)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concentrations. These results suggest that flavonoids reduce cytochrome c and that PBE inhibits electron transport chain activity mainly through NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases.

  4. Molecular basis of axonal dysfunction and traffic impairments in CMT.

    PubMed

    Gentil, Benoit J; Cooper, Laura

    2012-08-01

    Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. It comprises a group of diseases caused by mutations in genes involved in Schwann cells homeostasis and neuronal function that affect the peripheral nerves. So far mutations in more than 33 genes have been identified causing either the demyelinating form (CMT1) or the axonal form (CMT2). Genes involving a large variety of unrelated functions may lead to the same phenotype when mutated. Our review will focus on the common link between genes causing axonal phenotypes like MFN2, KIF1B, DYNC1H1, Rab7, TRPV4, ARSs, NEFL, HSPB1, MPZ, and HSPB8. While KIF1B and DYNC1H1, two genes coding for molecular motors, are directly linked to axonal transport, the involvement of the other CMT2-causing genes in this function is less obvious. However, the last years have seen a growing list of evidence demonstrating that intracellular trafficking and mitochondrial dynamics might be dysfunctional in CMT2, and these mechanisms might present a common link between dissimilar CMT2-causing genes. The involvement of impaired transport in the pathogenesis of other rare neurological diseases or recessive CMT2 is also discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Preserve and protect: maintaining axons within functional circuits

    PubMed Central

    Pease, Sarah E.; Segal, Rosalind A.

    2014-01-01

    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

  6. 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.

  7. Mitochondrial localization of P-glycoprotein and peptide transporters in corneal epithelial cells--novel strategies for intracellular drug targeting.

    PubMed

    Barot, Megha; Gokulgandhi, Mitan R; Pal, Dhananjay; Mitra, Ashim K

    2013-01-01

    This study was designed to investigate functional localization of both efflux (P-glycoprotein, P-gp) and influx (peptide) transporters in the mitochondrial membrane of cultured rabbit primary corneal epithelial cells (rPCECs). Isolation and purification of mitochondria was performed by optimized cell fractionation method. Mitochondrial integrity was measured by JC-1 uptake experiment. The efflux activity of P-gp was assessed by performing in vitro uptake studies on isolated mitochondria with Rhodamine 123 (Rho-123) alone and in the presence of P-gp inhibitors (quinidine and cyclosporine A) using fluorimetry and flow cytometry analysis. Functional activity of peptide transporter was assessed by performing in vitro uptake studies of [3H] Gly-sar on isolated mitochondria in the presence or absence of peptide transporter substrate (Val-Val). Molecular characterization of P-gp and peptide transporter was assessed by western blot and confocal analysis. Enhanced JC-1 accumulation in the isolated fraction confirmed mitochondrial membrane integrity. Significantly higher uptake of Rho-123 on isolated mitochondria was observed in the presence of quinidine (75 and 100 μM) and cyclosporine A (10 μM). Significantly lower uptake of [3H] Gly-sar was observed in the presence of val-val due to competitive inhibition of peptide transporter on isolated mitochondria. Western blot and confocal analysis further confirmed the presence of P-gp and peptide transporter on the mitochondrial membrane of rPCECs. The present study demonstrates the functional and molecular characterization of P-gp and peptide transporters in the mitochondrial membranes of rPCECs. This knowledge of mitochondrial existence of P-gp and peptide transporter will aid in the development of subcellular ocular drug delivery strategies.

  8. Green tea polyphenols require the mitochondrial iron transporter, mitoferrin, for lifespan extension in Drosophila melanogaster.

    PubMed

    Lopez, Terry E; Pham, Hoang M; Nguyen, Benjamin V; Tahmasian, Yerazik; Ramsden, Shannon; Coskun, Volkan; Schriner, Samuel E; Jafari, Mahtab

    2016-12-01

    Green tea has been found to increase the lifespan of various experimental animal models including the fruit fly, Drosophila melanogaster. High in polyphenolic content, green tea has been shown to reduce oxidative stress in part by its ability to bind free iron, a micronutrient that is both essential for and toxic to all living organisms. Due to green tea's iron-binding properties, we questioned whether green tea acts to increase the lifespan of the fruit fly by modulating iron regulators, specifically, mitoferrin, a mitochondrial iron transporter, and transferrin, found in the hemolymph of flies. Publicly available hypomorph mutants for these iron regulators were utilized to investigate the effect of green tea on lifespan and fertility. We identified that green tea could not increase the lifespan of mitoferrin mutants but did rescue the reduced male fertility phenotype. The effect of green tea on transferrin mutant lifespan and fertility were comparable to w(1118) flies, as observed in our previous studies, in which green tea increased male fly lifespan and reduced male fertility. Expression levels in both w(1118) flies and mutant flies, supplemented with green tea, showed an upregulation of mitoferrin but not transferrin. Total body and mitochondrial iron levels were significantly reduced by green tea supplementation in w(1118) and mitoferrin mutants but not transferrin mutant flies. Our results demonstrate that green tea may act to increase the lifespan of Drosophila in part by the regulation of mitoferrin and reduction of mitochondrial iron.

  9. Dicoumarol impairs mitochondrial electron transport and pyrimidine biosynthesis in human myeloid leukemia HL-60 cells.

    PubMed

    González-Aragón, David; Ariza, Julia; Villalba, José M

    2007-02-01

    Dicoumarol, a competitive inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), increases intracellular superoxide and affects cell growth of tumor cells. This work was set to establish a mechanistic link between dicoumarol, superoxide and cell cycle alterations in HL-60 cells. Using ES936, a mechanism-based irreversible inhibitor of NQO1, we demonstrate that NQO1 inhibition is not a major factor involved in superoxide boost. Mitochondrial Complexes II, III and IV were directly inhibited by dicoumarol. Succinate, which inhibits superoxide generation by reversed electron flow in Complex II, significantly decreased superoxide boost in dicoumarol-treated cells and in isolated mitochondria incubated with dicoumarol and decylubiquinol. Superoxide generation in cells was strongly potentiated by blocking the quinone site of Complex II with thenoyltrifluoroacetone, supporting the involvement of cytochrome b560 to drive electrons for increasing superoxide. Simultaneous inhibition of the mitochondrial chain upstream ubiquinone and displacement of succinate from the Complex II active site is proposed as a major mechanism to explain how dicoumarol increases superoxide in HL-60 cells. Dicoumarol-treated cells accumulated in S phase due to the impairment of pyrimidine biosynthesis at dihydroorotate dehydrogenase step because blockade was overcome by addition of exogenous uridine or orotate, but not by dihydroorotate. We demonstrate for the first time that dicoumarol inhibits mitochondrial electron transport, induces superoxide release by reversed electron flow in Complex II, and inhibits pyrimidines biosynthesis. These actions must be taken into account when considering dicoumarol effects on cells.

  10. Supercomplexes of the mitochondrial electron transport chain decline in the aging rat heart.

    PubMed

    Gómez, Luis A; Monette, Jeffrey S; Chavez, Juan D; Maier, Claudia S; Hagen, Tory M

    2009-10-01

    Accumulation of mitochondrial electron transport chain (ETC) defects is a recognized hallmark of the age-associated decline in cardiac bioenergetics; however, the molecular events involved are only poorly understood. In the present work, we hypothesized that age-related ETC deterioration stemmed partly from disassociation of large solid-state macromolecular assemblies termed "supercomplexes". Mitochondrial proteins from young and old rat hearts were separated by blue native-PAGE, protein bands analyzed by LC-MALDI-MS/MS, and protein levels quantified by densitometry. Results showed that supercomplexes comprised of various stoichiometries of complexes I, III and IV were observed, and declined significantly (p<0.05, n=4) with age. Supercomplexes displaying the highest molecular masses were the most severely affected. Considering that certain diseases (e.g. Barth Syndrome) display similar supercomplex destabilization as our results for aging, the deterioration in ETC supercomplexes may be an important underlying factor for both impaired mitochondrial function and loss of cardiac bioenergetics with age.

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

    PubMed

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

    2016-04-08

    A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state. Copyright © 2016, American Association for the Advancement of Science.

  12. Mitochondrial Lipid Abnormality and Electron Transport Chain Impairment in Mice Lacking α-Synuclein†

    PubMed Central

    Ellis, Christopher E.; Murphy, Eric J.; Mitchell, Drake C.; Golovko, Mikhail Y.; Scaglia, Fernando; Barceló-Coblijn, Gwendolyn C.; Nussbaum, Robert L.

    2005-01-01

    The presynaptic protein α-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking α-synuclein (Snca−/−), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca−/− brain suggest a relationship between α-synuclein's role in brain lipid metabolism, mitochondrial function, and PD. PMID:16260631

  13. Evolution of interacting proteins in the mitochondrial electron transport system in a marine copepod.

    PubMed

    Willett, Christopher S; Burton, Ronald S

    2004-03-01

    The extensive interaction between mitochondrial-encoded and nuclear-encoded subunits of electron transport system (ETS) enzymes in mitochondria is expected to lead to intergenomic coadaptation. Whether this coadaptation results from adaptation to the environment or from fixation of deleterious mtDNA mutations followed by compensatory nuclear gene evolution is unknown. The intertidal copepod Tigriopus californicus shows extreme divergence in mtDNA sequence and provides an excellent model system for study of intergenomic coadaptation. Here, we examine genes encoding subunits of complex III of the ETS, including the mtDNA-encoded cytochrome b (CYTB), the nuclear-encoded rieske iron-sulfur protein (RISP), and cytochrome c(1) (CYC1). We compare levels of polymorphism within populations and divergence between populations in these genes to begin to untangle the selective forces that have shaped evolution in these genes. CYTB displays dramatic divergence between populations, but sequence analysis shows no evidence for positive selection driving this divergence. CYC1 and RISP have lower levels of sequence divergence between populations than CYTB, but, again, sequence analysis gives no evidence for positive selection acting on them. However, an examination of variation at cytochrome c (CYC), a nuclear-encoded protein that transfers electrons between complex III and complex IV provides evidence for selective divergence. Hence, it appears that rapid evolution in mitochondrial-encoded subunits is not always associated with rapid divergence in interacting subunits (CYC1 and RISP), but can be in some cases (CYC). Finally, a comparison of nuclear-encoded and mitochondrial-encoded genes from T. californicus suggests that substitution rates in the mitochondrial-encoded genes are dramatically increased relative to nuclear genes.

  14. 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

  15. 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.

  16. Mitochondrial-Driven Bicarbonate Transport Supports Photosynthesis in a Marine Microalga1

    PubMed Central

    Huertas, I. Emma; Colman, Brian; Espie, George S.

    2002-01-01

    The CO2-concentrating mechanism (CCM) of the marine eustigmatophycean microalga Nannochloropsis gaditana consists of an active HCO3− transport system and an internal carbonic anhydrase to facilitate accumulation and conversion of HCO3− to CO2 for photosynthetic fixation. Aqueous inlet mass spectrometry revealed that a portion of the CO2 generated within the cells leaked to the medium, resulting in a significant rise in the extracellular CO2 concentration to a level above its chemical equilibrium that was diagnostic for active HCO3− transport. The transient rise in extracellular CO2 occurred in the light and the dark and was resolved from concurrent respiratory CO2 efflux using H13CO3− stable isotope techniques. H13CO3− pump-13CO2 leak activity of the CCM was unaffected by 10 μm 3(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of chloroplast linear electron transport, although photosynthetic O2 evolution was reduced by 90%. However, low concentrations of cyanide, azide, and rotenone along with anoxia significantly reduced or abolished 13CO2 efflux in the dark and light. These results indicate that H13CO3− transport was supported by