The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Christopher C. J. Miller - One of the best experts on this subject based on the ideXlab platform.
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Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of Neurofilaments.
European journal of cell biology, 2009Co-Authors: Darran Yates, Christopher Shaw, Catherine Manser, Kurt J. De Vos, Declan M. Mcloughlin, Christopher C. J. MillerAbstract: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.
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Neurofilament heavy chain side arm phosphorylation regulates axonal transport of Neurofilaments
Journal of Cell Biology, 2003Co-Authors: Steven Ackerley, Paul Thornhill, Nigel P Leigh, Janet Brownlees, Andrew J Grierson, Brian H. Anderton, C. Shaw, Christopher C. J. MillerAbstract:Neurofilaments possess side arms that comprise the carboxy-terminal domains of Neurofilament middle and heavy chains (NFM and NFH); that of NFH is heavily phosphorylated in axons. Here, we demonstrate that phosphorylation of NFH side arms is a mechanism for regulating transport of Neurofilaments through axons. Mutants in which known NFH phosphorylation sites were mutated to preclude phosphorylation or mimic permanent phosphorylation display altered rates of transport in a bulk transport assay. Similarly, application of roscovitine, an inhibitor of the NFH side arm kinase Cdk5/p35, accelerates Neurofilament transport. Analyses of Neurofilament movement in transfected living neurons demonstrated that a mutant mimicking permanent phosphorylation spent a higher proportion of time pausing than one that could not be phosphorylated. Thus, phosphorylation of NFH slows Neurofilament transport, and this is due to increased pausing in Neurofilament movement.
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Neurofilaments and neurological disease
BioEssays : news and reviews in molecular cellular and developmental biology, 2003Co-Authors: Ammar Al-chalabi, Christopher C. J. MillerAbstract:Neurofilaments are one of the major components of the neuronal cytoskeleton and are responsible for maintaining the calibre of axons. They are modified by post-translational changes that are regulated in complex fashions including by the interaction with neighbouring glial cells. Neurofilament accumulations are seen in several neurological diseases and Neurofilament mutations have now been associated with Charcot-Marie-Tooth disease, Parkinson's disease and amyotrophic lateral sclerosis. In this review, we discuss the structure, normal function and molecular pathology of Neurofilaments.
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Charcot-Marie-Tooth disease Neurofilament mutations disrupt Neurofilament assembly and axonal transport.
Human molecular genetics, 2002Co-Authors: Janet Brownlees, Steven Ackerley, Andrew J Grierson, Brian H. Anderton, P. Nigel Leigh, Christopher Shaw, Nick J.o. Jacobsen, Kerry Shea, Christopher C. J. MillerAbstract:Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system, and mutations in Neurofilaments have been linked to some forms of CMT. Neurofilaments are the major intermediate filaments of neurones, but the mechanisms by which the CMT mutations induce disease are not known. Here, we demonstrate that CMT mutant Neurofilaments disrupt both Neurofilament assembly and axonal transport of Neurofilaments in cultured mammalian cells and neurones. We also show that CMT mutant Neurofilaments perturb the localization of mitochondria in neurones. Accumulations of Neurofilaments are a pathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, and diabetic neuropathy. Our results demonstrate that aberrant Neurofilament assembly and transport can induce neurological disease, and further implicate defective Neurofilament metabolism in the pathogenesis of human neurodegenerative diseases.
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Axonal transport of Neurofilaments in normal and disease states
Cellular and molecular life sciences : CMLS, 2002Co-Authors: Christopher C. J. Miller, Janet Brownlees, Steven Ackerley, Andrew J Grierson, N. Jacobsen, P. ThornhillAbstract:Neurofilaments are among the most abundant organelles in neurones. They are synthesised in cell bodies and then transported into and through axons by a process termed 'slow axonal transport' at a rate that is distinct from that driven by conventional fast motors. Several recent studies have now demonstrated that this slow rate of transport is actually the consequence of conventional fast rates of movement that are interrupted by extended pausing. At any one time, most Neurofilaments are thus stationary. Accumulations of Neurofilaments are a pathological feature of several human neurodegenerative diseases suggesting that Neurofilament transport is disrupted in disease states. Here, we review recent advances in our understanding of Neurofilament transport in both normal and disease states. Increasing evidence suggests that phosphorylation of Neurofilaments is a mechanism for regulating their transport properties, possibly by promoting their detachment from the motor(s). In some neurodegenerative diseases, signal transduction mechanisms involving Neurofilament kinases and phosphatases may be perturbed leading to disruption of transport.
Aidong Yuan - One of the best experts on this subject based on the ideXlab platform.
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Peripherin Is a Subunit of Peripheral Nerve Neurofilaments: Implications for Differential Vulnerability of CNS and Peripheral Nervous System Axons
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2012Co-Authors: Aidong Yuan, Jean-pierre Julien, Mala V Rao, Ronald K.h. Liem, Takahiro Sasaki, Asok Kumar, Corrinne M. Peterhoff, Ralph A. NixonAbstract:Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the Neurofilament triplet proteins (NFL, NFM, and NFH) but has an unknown function. The earlier peak expression of peripherin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that peripherin and Neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, peripherin is as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry with these subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins in sciatic axons and co-localizes with NFL on single Neurofilament by immunogold electron microscopy. Peripherin also co-assembles into a single network of filaments containing NFL, NFM, NFH with and without α-internexin in quadruple- or quintuple-transfected SW13 vim (−) cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons. Moreover, peripherin mutations has been shown to disrupt the Neurofilament network in transfected SW13 vim(−) cells. These data show that peripherin and the Neurofilament proteins are functionally interdependent. The results strongly support the view that rather than forming an independent structure, peripherin is a subunit of Neurofilaments in the adult PNS. Our findings provide a basis for its close relationship with Neurofilaments in PNS diseases associated with Neurofilament accumulation.
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deleting the phosphorylated tail domain of the Neurofilament heavy subunit does not alter Neurofilament transport rate in vivo
Neuroscience Letters, 2006Co-Authors: Aidong Yuan, Ralph A. Nixon, Mala RaoAbstract:Abstract Phosphorylation of the carboxyl tail domains of the Neurofilament heavy (NF-H) and middle molecular weight (NF-M) subunits has been proposed to regulate the axonal transport of Neurofilaments. To test this hypothesis, we recently constructed mice lacking the extensively phosphorylated NF-H tail domain (NF-HtailΔ) and showed that the transport rate of Neurofilaments in optic axons is unaltered in the absence of this domain [M.V. Rao, M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.A. Calcutt, Y. Uchiyama, R.A. Nixon, D.W. Cleveland, Gene replacement in mice reveals that the heavily phosphorylated tail of Neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport, J. Cell Biol. 158 (2002) 681–693]. However, Shea et al. proposed that deletion of NF-H carboxyl-terminal region accelerates the transport of a subpopulation of Neurofilaments based on minor differences between tail-deleted and control mice in our axonal transport analysis. Here, we present additional evidence that Neurofilament transport rate is unchanged after deleting the phosphorylated NF-H tail domain, establishing unequivocally that the NF-H tail domain alone does not regulate the rate of Neurofilament transport in optic axons in vivo. Possible roles for tail domains as cross-bridges between a Neurofilament and its neighbors or other cytoskeletal elements is discussed.
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Deleting the phosphorylated tail domain of the Neurofilament heavy subunit does not alter Neurofilament transport rate in vivo.
Neuroscience letters, 2005Co-Authors: Aidong Yuan, Ralph A. Nixon, Mala V RaoAbstract:Phosphorylation of the carboxyl tail domains of the Neurofilament heavy (NF-H) and middle molecular weight (NF-M) subunits has been proposed to regulate the axonal transport of Neurofilaments. To test this hypothesis, we recently constructed mice lacking the extensively phosphorylated NF-H tail domain (NF-HtailDelta) and showed that the transport rate of Neurofilaments in optic axons is unaltered in the absence of this domain [M.V. Rao, M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.A. Calcutt, Y. Uchiyama, R.A. Nixon, D.W. Cleveland, Gene replacement in mice reveals that the heavily phosphorylated tail of Neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport, J. Cell Biol. 158 (2002) 681-693]. However, Shea et al. proposed that deletion of NF-H carboxyl-terminal region accelerates the transport of a subpopulation of Neurofilaments based on minor differences between tail-deleted and control mice in our axonal transport analysis. Here, we present additional evidence that Neurofilament transport rate is unchanged after deleting the phosphorylated NF-H tail domain, establishing unequivocally that the NF-H tail domain alone does not regulate the rate of Neurofilament transport in optic axons in vivo. Possible roles for tail domains as cross-bridges between a Neurofilament and its neighbors or other cytoskeletal elements is discussed.
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myosin va binding to Neurofilaments is essential for correct myosin va distribution and transport and Neurofilament density
Journal of Cell Biology, 2002Co-Authors: Mala V Rao, Aidong Yuan, Linda J Engle, Panaiyur S Mohan, Dike Qiu, Anne M Cataldo, Linda HassingerAbstract:The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with Neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L–null mice lacking Neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H–null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of Neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating Neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased Neurofilament number and levels of Neurofilament proteins without altering axon caliber. These results identify myosin Va as a Neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of Neurofilaments in axons.
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gene replacement in mice reveals that the heavily phosphorylated tail of Neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport
Journal of Cell Biology, 2002Co-Authors: Mala Rao, Salvatore Mattina, Christopher M. Ward, Aidong Yuan, Michael L Garcia, Yukio Miyazaki, Nigel A Calcutt, Yasuo Uchiyama, Takahiro Gotow, Ralph A. NixonAbstract:The COOH-terminal tail of mammalian Neurofilament heavy subunit (NF-H), the largest Neurofilament subunit, contains 44-51 lysine–serine–proline repeats that are nearly stoichiometrically phosphorylated after assembly into Neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between Neurofilaments or from Neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of Neurofilaments, alter the ratios of the three Neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most Neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of Neurofilament subunits.
Jean-pierre Julien - One of the best experts on this subject based on the ideXlab platform.
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Local Acceleration of Neurofilament Transport at Nodes of Ranvier.
The Journal of Neuroscience, 2018Co-Authors: Cynthia L. Walker, Peter Jung, Atsuko Uchida, Niraj Trivedi, J. Daniel Fenn, Paula C. Monsma, Roxanne Larivière, Jean-pierre Julien, Anthony BrownAbstract:Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are Neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in Neurofilament number that can be as much as 10-fold in the largest axons. To investigate how Neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent Neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze Neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo. Fluorescent Neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that Neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of Neurofilament transport (i.e., the proportion of the time that the Neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant Neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, Neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable Neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains. SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in Neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of Neurofilaments, which move along axons in a rapid intermittent manner. Imaging of Neurofilament transport in mature myelinated axons ex vivo reveals that Neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to Neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.
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Peripherin Is a Subunit of Peripheral Nerve Neurofilaments: Implications for Differential Vulnerability of CNS and Peripheral Nervous System Axons
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2012Co-Authors: Aidong Yuan, Jean-pierre Julien, Mala V Rao, Ronald K.h. Liem, Takahiro Sasaki, Asok Kumar, Corrinne M. Peterhoff, Ralph A. NixonAbstract:Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the Neurofilament triplet proteins (NFL, NFM, and NFH) but has an unknown function. The earlier peak expression of peripherin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that peripherin and Neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, peripherin is as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry with these subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins in sciatic axons and co-localizes with NFL on single Neurofilament by immunogold electron microscopy. Peripherin also co-assembles into a single network of filaments containing NFL, NFM, NFH with and without α-internexin in quadruple- or quintuple-transfected SW13 vim (−) cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons. Moreover, peripherin mutations has been shown to disrupt the Neurofilament network in transfected SW13 vim(−) cells. These data show that peripherin and the Neurofilament proteins are functionally interdependent. The results strongly support the view that rather than forming an independent structure, peripherin is a subunit of Neurofilaments in the adult PNS. Our findings provide a basis for its close relationship with Neurofilaments in PNS diseases associated with Neurofilament accumulation.
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New movements in Neurofilament transport, turnover and disease.
Experimental cell research, 2007Co-Authors: Devin M. Barry, Jean-pierre Julien, Stéphanie Millecamps, Michael L GarciaAbstract:Revealing the mechanisms by which Neurofilament transport and turnover are regulated has proven difficult over the years but recent studies have given new insight into these processes. Mature Neurofilament fibers may incorporate a fourth functional subunit, α-internexin, as new evidence suggests. Recent findings have made the role of phosphorylation in regulating Neurofilament transport velocity controversial. Kinesin and dynein may transport Neurofilaments in slow axonal transport as they have been found to associate with Neurofilaments. Neurofilament transport and turnover rates may be reduced depending on the existing stationary Neurofilament network. Finally, mutations in Neurofilament light that have been linked to Charcot-Marie-Tooth disease as well as other Neurofilament abnormalities in human disease are discussed.
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Interactions between peripherin and Neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 1999Co-Authors: Jean-martin Beaulieu, Janice Robertson, Jean-pierre JulienAbstract:Neurofilaments are the principal intermediate filament type expressed by neurons. They are formed by the co-assembly of three subunits: NF-L, NF-M, and NF-H. Peripherin is another intermediate filament protein expressed mostly in neurons of the peripheral nervous system. In contrast to Neurofilaments, peripherin can self-assemble to establish an intermediate filament network in cultured cells. The co-expression of Neurofilaments and peripherin is found mainly during development and regeneration. We used SW13 cells devoid of endogenous cytoplasmic intermediate filaments to assess the exact assembly characteristics of peripherin with each Neurofilament subunit. Our results demonstrate that peripherin can assemble with NF-L. In contrast, the co-expression of peripherin with the large Neurofilament subunits interferes with peripherin assembly. These results confirm the existence of interactions between peripherin and Neurofilaments in physiological conditions. Moreover, they suggest that perturbations in the stoichiometry of Neurofilaments can have an impact on peripherin assembly in vivo.
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Neurofilament functions in health and disease.
Current opinion in neurobiology, 1999Co-Authors: Jean-pierre JulienAbstract:Abstract Transgenic approaches have recently been used to investigate the functions of neuronal intermediate filaments. Gene knockout studies have demonstrated that Neurofilaments are not required for axogenesis and that individual Neurofilament proteins play distinct roles in filament assembly and in the radial growth of axons. The involvement of Neurofilaments in disease is supported by the discovery of novel mutations in the Neurofilament heavy gene from cases of amyotrophic lateral sclerosis and by reports of neuronal death in mouse models expressing Neurofilament and α-internexin transgenes. However, mouse studies have shown that axonal Neurofilaments are not required for pathogenesis caused by mutations in superoxide dismutase and that increasing perikaryal levels of Neurofilament proteins may even confer protection in this disease.
Mala V Rao - One of the best experts on this subject based on the ideXlab platform.
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Peripherin Is a Subunit of Peripheral Nerve Neurofilaments: Implications for Differential Vulnerability of CNS and Peripheral Nervous System Axons
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2012Co-Authors: Aidong Yuan, Jean-pierre Julien, Mala V Rao, Ronald K.h. Liem, Takahiro Sasaki, Asok Kumar, Corrinne M. Peterhoff, Ralph A. NixonAbstract:Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the Neurofilament triplet proteins (NFL, NFM, and NFH) but has an unknown function. The earlier peak expression of peripherin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that peripherin and Neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, peripherin is as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry with these subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins in sciatic axons and co-localizes with NFL on single Neurofilament by immunogold electron microscopy. Peripherin also co-assembles into a single network of filaments containing NFL, NFM, NFH with and without α-internexin in quadruple- or quintuple-transfected SW13 vim (−) cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons. Moreover, peripherin mutations has been shown to disrupt the Neurofilament network in transfected SW13 vim(−) cells. These data show that peripherin and the Neurofilament proteins are functionally interdependent. The results strongly support the view that rather than forming an independent structure, peripherin is a subunit of Neurofilaments in the adult PNS. Our findings provide a basis for its close relationship with Neurofilaments in PNS diseases associated with Neurofilament accumulation.
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Deleting the phosphorylated tail domain of the Neurofilament heavy subunit does not alter Neurofilament transport rate in vivo.
Neuroscience letters, 2005Co-Authors: Aidong Yuan, Ralph A. Nixon, Mala V RaoAbstract:Phosphorylation of the carboxyl tail domains of the Neurofilament heavy (NF-H) and middle molecular weight (NF-M) subunits has been proposed to regulate the axonal transport of Neurofilaments. To test this hypothesis, we recently constructed mice lacking the extensively phosphorylated NF-H tail domain (NF-HtailDelta) and showed that the transport rate of Neurofilaments in optic axons is unaltered in the absence of this domain [M.V. Rao, M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.A. Calcutt, Y. Uchiyama, R.A. Nixon, D.W. Cleveland, Gene replacement in mice reveals that the heavily phosphorylated tail of Neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport, J. Cell Biol. 158 (2002) 681-693]. However, Shea et al. proposed that deletion of NF-H carboxyl-terminal region accelerates the transport of a subpopulation of Neurofilaments based on minor differences between tail-deleted and control mice in our axonal transport analysis. Here, we present additional evidence that Neurofilament transport rate is unchanged after deleting the phosphorylated NF-H tail domain, establishing unequivocally that the NF-H tail domain alone does not regulate the rate of Neurofilament transport in optic axons in vivo. Possible roles for tail domains as cross-bridges between a Neurofilament and its neighbors or other cytoskeletal elements is discussed.
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myosin va binding to Neurofilaments is essential for correct myosin va distribution and transport and Neurofilament density
Journal of Cell Biology, 2002Co-Authors: Mala V Rao, Aidong Yuan, Linda J Engle, Panaiyur S Mohan, Dike Qiu, Anne M Cataldo, Linda HassingerAbstract:The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with Neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L–null mice lacking Neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H–null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of Neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating Neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased Neurofilament number and levels of Neurofilament proteins without altering axon caliber. These results identify myosin Va as a Neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of Neurofilaments in axons.
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Neurofilament-dependent radial growth of motor axons and axonal organization of Neurofilaments does not require the Neurofilament heavy subunit (NF-H) or its phosphorylation.
The Journal of cell biology, 1998Co-Authors: Mala V Rao, Megan K. Houseweart, Toni L. Williamson, Thomas O. Crawford, Janet Folmer, Don W ClevelandAbstract:Neurofilaments are essential for establishment and maintenance of axonal diameter of large myelinated axons, a property that determines the velocity of electrical signal conduction. One prominent model for how Neurofilaments specify axonal growth is that the 660–amino acid, heavily phosphorylated tail domain of Neurofilament heavy subunit (NF-H) is responsible for Neurofilament-dependent structuring of axoplasm through intra-axonal crossbridging between adjacent Neurofilaments or to other axonal structures. To test such a role, homologous recombination was used to generate NF-H–null mice. In peripheral motor and sensory axons, absence of NF-H does not significantly affect the number of Neurofilaments or axonal elongation or targeting, but it does affect the efficiency of survival of motor and sensory axons. Loss of NF-H caused only a slight reduction in nearest neighbor spacing of Neurofilaments and did not affect Neurofilament distribution in either large- or small-diameter motor axons. Since postnatal growth of motor axon caliber continues largely unabated in the absence of NF-H, neither interactions mediated by NF-H nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.
Michael L Garcia - One of the best experts on this subject based on the ideXlab platform.
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Genetic Manipulation of Neurofilament Protein Phosphorylation
Methods in enzymology, 2015Co-Authors: Maria R. Jones, Eric Villalón, Michael L GarciaAbstract:Neurofilament biology is important to understanding structural properties of axons, such as establishment of axonal diameter by radial growth. In order to study the function of Neurofilaments, a series of genetically modified mice have been generated. Here, we describe a brief history of genetic modifications used to study Neurofilaments, as well as an overview of the steps required to generate a gene-targeted mouse. In addition, we describe steps utilized to analyze Neurofilament phosphorylation status using immunoblotting. Taken together, these provide comprehensive analysis of Neurofilament function in vivo, which can be applied to many systems.
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Distal to proximal development of peripheral nerves requires the expression of Neurofilament heavy
Neuroscience, 2010Co-Authors: Hailian Shen, Devin M. Barry, Michael L GarciaAbstract:At the initiation of radial growth, Neurofilaments are likely to consist primarily of Neurofilament light and medium as Neurofilament heavy expression is developmentally delayed. To better understand the role of Neurofilament heavy in structuring axons, axonal diameter and Neurofilament organization were measured in proximal and distal segments of the sciatic nerve and along the entire length of the phrenic nerve. Deletion of Neurofilament heavy reduced axonal diameters and Neurofilament number in proximal nerve segments. However, Neurofilament spacing was greater in proximal versus distal phrenic nerve segments. Taken together, these results suggest that loss of Neurofilament heavy reduces radial growth in proximal axonal segments by reducing the accumulation of Neurofilaments. As Neurofilament heavy expression is developmentally delayed, these results suggest that without Neurofilament heavy, the Neurofilament network is established in a distal to proximal gradient perhaps to allow distal axonal segments to develop prior to proximal segments.
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New movements in Neurofilament transport, turnover and disease.
Experimental cell research, 2007Co-Authors: Devin M. Barry, Jean-pierre Julien, Stéphanie Millecamps, Michael L GarciaAbstract:Revealing the mechanisms by which Neurofilament transport and turnover are regulated has proven difficult over the years but recent studies have given new insight into these processes. Mature Neurofilament fibers may incorporate a fourth functional subunit, α-internexin, as new evidence suggests. Recent findings have made the role of phosphorylation in regulating Neurofilament transport velocity controversial. Kinesin and dynein may transport Neurofilaments in slow axonal transport as they have been found to associate with Neurofilaments. Neurofilament transport and turnover rates may be reduced depending on the existing stationary Neurofilament network. Finally, mutations in Neurofilament light that have been linked to Charcot-Marie-Tooth disease as well as other Neurofilament abnormalities in human disease are discussed.
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gene replacement in mice reveals that the heavily phosphorylated tail of Neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport
Journal of Cell Biology, 2002Co-Authors: Mala Rao, Salvatore Mattina, Christopher M. Ward, Aidong Yuan, Michael L Garcia, Yukio Miyazaki, Nigel A Calcutt, Yasuo Uchiyama, Takahiro Gotow, Ralph A. NixonAbstract:The COOH-terminal tail of mammalian Neurofilament heavy subunit (NF-H), the largest Neurofilament subunit, contains 44-51 lysine–serine–proline repeats that are nearly stoichiometrically phosphorylated after assembly into Neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between Neurofilaments or from Neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of Neurofilaments, alter the ratios of the three Neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most Neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of Neurofilament subunits.
