RESUMEN
Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament triplet proteins [neurofilament light (NFL), medium (NFM), and heavy (NFH) chain] 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 triplets form separate filament systems. However, here, we demonstrate 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 colocalizes with NFL on single neurofilaments by immunogold electron microscopy. Peripherin also coassembles into a single network of filaments containing NFL, NFM, and NFH with and without α-internexin in quadruple- or quintuple-transfected SW13vim(-) 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 SW13vim(-) 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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Axones/metabolismo , Sistema Nervioso Central/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Sistema Nervioso Periférico/metabolismo , Animales , Anticuerpos Monoclonales , Axones/ultraestructura , Western Blotting , Sistema Nervioso Central/citología , Sistema Nervioso Central/ultraestructura , Electroforesis en Gel de Poliacrilamida , Femenino , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Sistema Nervioso Periférico/citología , Sistema Nervioso Periférico/ultraestructura , Periferinas , Nervio Ciático/citología , Nervio Ciático/metabolismo , TransfecciónRESUMEN
Neurofilament and tau proteins are neuron-specific cytoskeletal proteins that are enriched in axons, regulated by many of the same protein kinases, interact physically, and are the principal constituents of neurofibrillary lesions in major adult-onset dementias. Both proteins share functions related to the modulation of stability and functions of the microtubule network in axons, axonal transport and scaffolding of organelles, long-term synaptic potentiation, and learning and memory. Expression of these proteins is regulated not only at the transcriptional level but also through posttranscriptional control of pre-mRNA splicing, mRNA stability, transport, localization, local translation and degradation. Current evidence suggests that posttranscriptional determinants of their levels are usually regulated by RNA-binding proteins and microRNAs primarily through 3'-untranslated regions of neurofilament and tau mRNAs. Dysregulations of neurofilament and tau expression caused by mutations or pathologies of RNA-binding proteins such as TDP43, FUS and microRNAs are increasingly recognized in association with varied neurological disorders. In this review, we summarize the current understanding of posttranscriptional control of neurofilament and tau by examining the posttranscriptional regulation of neurofilament and tau by RNA-binding proteins and microRNAs implicated in health and diseases.
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MicroARNs , Proteínas de Neurofilamentos , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Axones/metabolismo , Transporte Axonal , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.
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Autofagia , Filamentos Intermedios , Ratones , Animales , Autofagia/fisiología , Proteolisis , Filamentos Intermedios/metabolismo , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismoRESUMEN
OBJECTIVE: This study was conducted to investigate the effect of long non-coding RNA (lncRNA) Gm37494 on osteoarthritis (OA) and its related molecular mechanism. METHODS: The cartilage tissues were obtained from OA patients, and an OA mouse model was induced by the destabilization of the medial meniscus, followed by measurement of Gm37494, microRNA (miR)-181a-5p, GABRA1 mRNA, and the encoded GABAARα1 protein expression. Thereafter, a cellular model was induced by interleukin-1ß (IL-1ß) treatment in chondrocytes, followed by ectopic and silencing experiments. Chondrocyte proliferation was detected by CCK-8 and EdU assays, chondrocyte apoptosis by flow cytometry and western blot, and the levels of inflammatory factors by ELISA. The binding of Gm37494 to miR-181a-5p was evaluated by dual-luciferase reporter gene and RIP assays, and that of GABRA1 to miR-181a-5p by dual-luciferase reporter gene and RNA pull-down assays. RESULTS: OA patients and mice had decreased GABRA1 mRNA and GABAARα1 protein levels and elevated miR-181a-5p expression in cartilage tissues. Additionally, Gm37494 was poorly expressed in OA mice. Mechanistically, Gm37494 directly bound to and inversely modulated miR-181a-5p that negatively targeted GABRA1. In IL-1ß-induced chondrocytes, Gm37494 overexpression enhanced cell proliferation and suppressed cell apoptosis and inflammation, whereas further miR-181a-5p up-regulation or GABRA1 silencing abolished these trends. CONCLUSIONS: Conclusively, Gm37494 elevated GABRA1 expression by binding to miR-181a-5p, thus ameliorating OA-induced chondrocyte damage.
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MicroARNs , Osteoartritis , ARN Largo no Codificante , Animales , Apoptosis/genética , Condrocitos/metabolismo , Regulación hacia Abajo , Humanos , Interleucina-1beta/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Osteoartritis/genética , Osteoartritis/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Ácido gamma-AminobutíricoRESUMEN
Biomarkers of neurodegeneration and neuronal injury have the potential to improve diagnostic accuracy, disease monitoring, prognosis, and measure treatment efficacy. Neurofilament proteins (NfPs) are well suited as biomarkers in these contexts because they are major neuron-specific components that maintain structural integrity and are sensitive to neurodegeneration and neuronal injury across a wide range of neurologic diseases. Low levels of NfPs are constantly released from neurons into the extracellular space and ultimately reach the cerebrospinal fluid (CSF) and blood under physiological conditions throughout normal brain development, maturation, and aging. NfP levels in CSF and blood rise above normal in response to neuronal injury and neurodegeneration independently of cause. NfPs in CSF measured by lumbar puncture are about 40-fold more concentrated than in blood in healthy individuals. New ultra-sensitive methods now allow minimally invasive measurement of these low levels of NfPs in serum or plasma to track disease onset and progression in neurological disorders or nervous system injury and assess responses to therapeutic interventions. Any of the five Nf subunits - neurofilament light chain (NfL), neurofilament medium chain (NfM), neurofilament heavy chain (NfH), alpha-internexin (INA) and peripherin (PRPH) may be altered in a given neuropathological condition. In familial and sporadic Alzheimer's disease (AD), plasma NfL levels may rise as early as 22 years before clinical onset in familial AD and 10 years before sporadic AD. The major determinants of elevated levels of NfPs and degradation fragments in CSF and blood are the magnitude of damaged or degenerating axons of fiber tracks, the affected axon caliber sizes and the rate of release of NfP and fragments at different stages of a given neurological disease or condition directly or indirectly affecting central nervous system (CNS) and/or peripheral nervous system (PNS). NfPs are rapidly emerging as transformative blood biomarkers in neurology providing novel insights into a wide range of neurological diseases and advancing clinical trials. Here we summarize the current understanding of intracellular NfP physiology, pathophysiology and extracellular kinetics of NfPs in biofluids and review the value and limitations of NfPs and degradation fragments as biomarkers of neurodegeneration and neuronal injury.
RESUMEN
Magnetic resonance diffusion-weighted imaging (DTI) provides a unique perspective on the pathophysiological and microstructural changes during spinal cord injury, with high spatial specificity; meanwhile, NM reflects the conduction and integrity of neuroelectrical signals in spinal cord fiber tracts, with time-specific and dynamic evaluation effects. The fractional anisotropy (FA) value, SEP amplitude, and neurological function score or improvement rate are correlated. The combination of DTI and NM can more reliably quantify the spinal cord function, evaluate the effectiveness of treatment, and determine the patient's prognosis, which can provide reference for clinical decision making and future research for SCI patients. That is, the lower the preoperative FA value and the lower the SEP amplitude, the worse the preoperative and postoperative neurological function, the lower the improvement rate, and the worse the prognosis of patients. Therefore, we believe that spinal cord function can be graded according to JOA scores to find the corresponding FA and SEP amplitude ranges and that, by measuring FA and SEP amplitude in the future, we can reverse the assessment of spinal cord function, expected postoperative improvement, and long-term prognosis. At the same time, FA values can also help determine the nature of the lesion to some extent.
Asunto(s)
Imagen de Difusión Tensora , Hernia/etiología , Traumatismos de la Médula Espinal , Anisotropía , Humanos , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/diagnóstico por imagenRESUMEN
The ultrastructural view of the axonal cytoskeleton as an extensively cross-linked network of neurofilaments (NFs) and other cytoskeletal polymers contrasts with the dynamic view suggested by axonal transport studies on cytoskeletal elements. Here we reconcile these perspectives by showing that neurons form a large NF network along axons which is unequivocally stationary, metabolically stable, and maintained by NFs and nonfilamentous subunit assemblies undergoing slow transport by intermittent rapid movements and pauses. In mouse primary cortical neurons transfected with EGFP-NFL, formation of this stationary NF network requires a critical level of NFs, which explains its absence in NF-poor developing neurons studied previously. Most NFs at proximal axon regions were in a stationary structure coexisting with a smaller pool of moving EGFP-NFL assemblies that were mainly nonfilamentous. Distally along the same axon, EGFP-labeled NFL was much less abundant, and we detected only short filaments moving bidirectionally by slow transport (rapid movements and pauses) as previously described. In living mice, >25% of radiolabeled newly synthesized NFs remained in optic axons after slowly transported NFs had exited. Retained NF remained fixed over several months in a nonuniform distribution and exhibited exceptionally slow turnover (t(1/2) >2.5 months), implying that, at steady state, >90% of NFs in mature optic axons comprise the stationary cytoskeleton and <10% are undergoing slow transport. These findings reconcile in vitro and in vivo axonal transport observations, showing that slowly transported NFs or subunit oligomers are precursors to a highly stable stationary cytoskeletal network that supports mature axons.
Asunto(s)
Axones/fisiología , Citoesqueleto/fisiología , Filamentos Intermedios/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Axones/química , Células Cultivadas , Citoesqueleto/química , Filamentos Intermedios/química , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/análisis , RatasRESUMEN
White matter disorders can involve injury to myelin or axons but the respective contribution of each to clinical course is difficult to evaluate non-invasively. Here, to develop a paradigm for further investigations of axonal pathology by MRI, we compared two genetic mouse models exhibiting relatively selective axonal or myelin deficits using quantitative MRI relaxography of the transverse relaxation times (T2) in vivo and ultrastructural morphometry. In HM-DKO mice, which lack genes encoding the heavy (NF-H) and medium (NF-M) subunits of neurofilaments, neurofilament content of large myelinated axons of the central nervous system (CNS) is markedly reduced in the absence of changes in myelin thickness and volume. In shiverer mutant mice, which lack functional myelin basic protein, CNS myelin sheath formation is markedly reduced but neurofilament content is normal. We observed increases in T2 in nearly all white matter in shiverer mice compared to their wild type, while more subtle increases in T2 were observed in HM-DKO in the corpus callosum. White matter T2 was generally greater in shiverer mice than HM-DKO mice. Ultrastructural morphometry of the corpus callosum, which exhibited the greatest T2 differences, confirmed that total cross-sectional area occupied by axons was similar in the two mouse models and that the major ultrastructural differences, determined by morphometry, were an absence of myelin and larger unmyelinated axons in shiverer mice and absence of neurofilaments in HM-DKO mice. Our findings indicate that T2 is strongly influenced by myelination state and axonal volume, while neurofilament structure within the intra-axonal compartment has a lesser effect upon single compartment T2 estimates.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/patología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Proteína Básica de Mielina/metabolismo , Proteínas de Neurofilamentos/metabolismo , Animales , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes NeurológicosRESUMEN
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.
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Transporte Axonal/genética , Proteínas tau/metabolismo , Animales , Ratones , Ratones Noqueados , Ratones Transgénicos , Nervio Óptico/metabolismo , Nervio Óptico/ultraestructura , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/ultraestructura , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.
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Axones/metabolismo , División Celular/fisiología , Citoesqueleto/metabolismo , Proteínas de Neurofilamentos/metabolismo , Subunidades de Proteína/metabolismo , Animales , Transporte Axonal/fisiología , Axones/ultraestructura , Supervivencia Celular , Marcación de Gen , Ratones , Ratones Transgénicos , Microtúbulos/metabolismo , Peso Molecular , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/genética , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genéticaRESUMEN
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.
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Transporte Axonal/fisiología , Axones/ultraestructura , Microtúbulos/metabolismo , Proteínas de Neurofilamentos/metabolismo , Tubulina (Proteína)/metabolismo , Potenciales de Acción/fisiología , Sustitución de Aminoácidos , Animales , Axones/fisiología , División Celular/fisiología , Células Cultivadas , Eliminación de Gen , Ratones , Vaina de Mielina , Proteínas de Neurofilamentos/genética , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Fosforilación , Subunidades de Proteína , Secuencias Repetitivas de AminoácidoRESUMEN
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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Transporte Axonal/fisiología , Axones/fisiología , Miosina Tipo V/metabolismo , Proteínas de Neurofilamentos/metabolismo , Animales , Axones/química , Axones/ultraestructura , Bacterias , Citoesqueleto/metabolismo , Filamentos Intermedios/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Inmunoelectrónica , Proteínas Motoras Moleculares/metabolismo , Miosina Tipo V/análisis , Miosina Tipo V/genética , Proteínas de Neurofilamentos/análisis , Proteínas de Neurofilamentos/genética , Proteínas Recombinantes de Fusión/metabolismo , Nervio Ciático/metabolismoRESUMEN
Neurofilament (NFL) proteins have recently been found to play unique roles in synapses. NFL is known to interact with the GluN1 subunit of N-methyl-D-aspartic acid (NMDAR) and be reduced in schizophrenia though functional consequences are unknown. Here we investigated whether the interaction of NFL with GluN1 modulates synaptic transmission and schizophrenia-associated behaviors. The interaction of NFL with GluN1 was assessed by means of molecular, pharmacological, electrophysiological, magnetic resonance spectroscopy (MRS), and schizophrenia-associated behavior analyses. NFL deficits cause an NMDAR hypofunction phenotype including abnormal hippocampal function, as seen in schizophrenia. NFL-/- deletion in mice reduces dendritic spines and GluN1 protein levels, elevates ubiquitin-dependent turnover of GluN1 and hippocampal glutamate measured by MRS, and depresses hippocampal long-term potentiation. NMDAR-related behaviors are also impaired, including pup retrieval, spatial and social memory, prepulse inhibition, night-time activity, and response to NMDAR antagonist, whereas motor deficits are minimal. Importantly, partially lowering NFL in NFL+/- mice to levels seen regionally in schizophrenia, induced similar but milder NMDAR-related synaptic and behavioral deficits. Our findings support an emerging view that central nervous system neurofilament subunits including NFL in the present report, serve distinctive, critical roles in synapses relevant to neuropsychiatric diseases.
Asunto(s)
Espinas Dendríticas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteínas de Neurofilamentos/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Conducta Animal , Femenino , Ácido Glutámico/metabolismo , Hipocampo/patología , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones NoqueadosRESUMEN
Alpha-internexin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H) but has an unknown function. The earlier peak expression of alpha-internexin 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 alpha-internexin and neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, alpha-internexin is as abundant as the triplet in the adult CNS and exists in a relatively fixed stoichiometry with these subunits. Alpha-internexin exhibits transport and turnover rates identical to those of triplet proteins in optic axons and colocalizes with NF-M on single neurofilaments by immunogold electron microscopy. Alpha-internexin also coassembles with all three neurofilament proteins into a single network of filaments in quadruple-transfected SW13vim(-) cells. Genetically deleting NF-M alone or together with NF-H in mice dramatically reduces alpha-internexin transport and content in axons throughout the CNS. Moreover, deleting alpha-internexin potentiates the effects of NF-M deletion on NF-H and NF-L transport. Finally, overexpressing a NF-H-LacZ fusion protein in mice induces alpha-internexin and neurofilament triplet to aggregate in neuronal perikarya and greatly reduces their transport and content selectively in axons. Our data show that alpha-internexin and the neurofilament proteins are functionally interdependent. The results strongly support the view that alpha-internexin is a fourth subunit of neurofilaments in the adult CNS, providing a basis for its close relationship with neurofilaments in CNS diseases associated with neurofilament accumulation.
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Axones/química , Proteínas de Filamentos Intermediarios/fisiología , Filamentos Intermedios/química , Proteínas de Neurofilamentos/fisiología , Animales , Axones/ultraestructura , Cruzamientos Genéticos , Femenino , Proteínas de Filamentos Intermediarios/análisis , Proteínas de Filamentos Intermediarios/deficiencia , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/ultraestructura , Filamentos Intermedios/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Complejos Multiproteicos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Proteínas de Neurofilamentos/análisis , Proteínas de Neurofilamentos/deficiencia , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/ultraestructura , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/fisiología , Células Ganglionares de la Retina/química , Células Ganglionares de la Retina/ultraestructura , Médula Espinal/química , Médula Espinal/ultraestructura , Relación Estructura-Actividad , TransfecciónRESUMEN
SUMMARYNeurofilaments (NFs) are unique among tissue-specific classes of intermediate filaments (IFs) in being heteropolymers composed of four subunits (NF-L [neurofilament light]; NF-M [neurofilament middle]; NF-H [neurofilament heavy]; and α-internexin or peripherin), each having different domain structures and functions. Here, we review how NFs provide structural support for the highly asymmetric geometries of neurons and, especially, for the marked radial expansion of myelinated axons crucial for effective nerve conduction velocity. NFs in axons extensively cross-bridge and interconnect with other non-IF components of the cytoskeleton, including microtubules, actin filaments, and other fibrous cytoskeletal elements, to establish a regionally specialized network that undergoes exceptionally slow local turnover and serves as a docking platform to organize other organelles and proteins. We also discuss how a small pool of oligomeric and short filamentous precursors in the slow phase of axonal transport maintains this network. A complex pattern of phosphorylation and dephosphorylation events on each subunit modulates filament assembly, turnover, and organization within the axonal cytoskeleton. Multiple factors, and especially turnover rate, determine the size of the network, which can vary substantially along the axon. NF gene mutations cause several neuroaxonal disorders characterized by disrupted subunit assembly and NF aggregation. Additional NF alterations are associated with varied neuropsychiatric disorders. New evidence that subunits of NFs exist within postsynaptic terminal boutons and influence neurotransmission suggests how NF proteins might contribute to normal synaptic function and neuropsychiatric disease states.
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Filamentos Intermedios/fisiología , Proteínas de Neurofilamentos/fisiología , Animales , Biomarcadores/metabolismo , Humanos , Filamentos Intermedios/metabolismo , Trastornos Mentales/fisiopatología , Proteínas de Neurofilamentos/metabolismo , Orgánulos/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
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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Transporte Axonal/fisiología , Eliminación de Gen , Proteínas de Neurofilamentos/metabolismo , Subunidades de Proteína/metabolismo , Animales , Transporte Axonal/genética , Metionina/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/genética , Fosforilación , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Isótopos de Azufre/metabolismo , Factores de TiempoRESUMEN
Neurofilaments are uniquely complex among classes of intermediate filaments in being composed of four subunits (NFL, NFM, NFH and alpha-internexin in the CNS) that differ in structure, regulation, and function. Although neurofilaments have been traditionally viewed as axonal structural components, recent evidence has revealed that distinctive assemblies of neurofilament subunits are integral components of synapses, especially at postsynaptic sites. Within the synaptic compartment, the individual subunits differentially modulate neurotransmission and behavior through interactions with specific neurotransmitter receptors. These newly uncovered functions suggest that alterations of neurofilament proteins not only underlie axonopathy in various neurological disorders but also may play vital roles in cognition and neuropsychiatric diseases. Here, we review evidence that synaptic neurofilament proteins are a sizable population in the CNS and we advance the concept that changes in the levels or post-translational modification of individual NF subunits contribute to synaptic and behavioral dysfunction in certain neuropsychiatric conditions.
Asunto(s)
Trastornos Mentales/metabolismo , Proteínas de Neurofilamentos/metabolismo , Sinapsis/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Médula Espinal/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Neurofilament assembly requires at minimum the polymerization of neurofilament light chain (NF-L) with either neurofilament medium chain (NF-M) or neurofilament heavy chain (NF-H) subunits, but requirements for their axonal transport have long been controversial. Using a gene deletion approach, we generated mice containing only NF-L or NF-M. In vivo pulse radiolabeling analyses in retinal ganglion cell neurons revealed that NF-L alone is incapable of efficient transport, whereas nearly one-half of the normal level of NF-M is transported along optic axons in the absence of the other triplet subunits. Under these conditions, however, NF-M transport is completely abolished by deleting alpha-internexin. Our results strongly suggest that efficient neurofilament protein transport in vivo minimally requires hetero-oligomer formation. They also show that NF-M can partner with intermediate filament proteins other than the NF-H and NF-L subunits in neurons to support slow transport and possibly other functions of neuronal intermediate filaments.
Asunto(s)
Proteínas de Neurofilamentos/metabolismo , Animales , Transporte Axonal/fisiología , Axones/metabolismo , Axones/ultraestructura , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Femenino , Proteínas de Filamentos Intermediarios , Filamentos Intermedios/metabolismo , Filamentos Intermedios/ultraestructura , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neurofilamentos/deficiencia , Proteínas de Neurofilamentos/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
The axonal cytoskeleton of neurofilament (NF) is a long-lived network of fibrous elements believed to be a stationary structure maintained by a small pool of transported cytoskeletal precursors. Accordingly, it may be predicted that NF content in axons can vary independently from the transport rate of NF. In the present report, we confirm this prediction by showing that human NFH transgenic mice and transgenic mice expressing human NFL Ser55 (Asp) develop nearly identical abnormal patterns of NF accumulation and distribution in association with opposite changes in NF slow transport rates. We also show that the rate of NF transport in wild-type mice remains constant along a length of the optic axon where NF content varies 3-fold. Moreover, knockout mice lacking NFH develop even more extreme (6-fold) proximal to distal variation in NF number, which is associated with a normal wild-type rate of NF transport. The independence of regional NF content and NF transport is consistent with previous evidence suggesting that the rate of incorporation of transported NF precursors into a metabolically stable stationary cytoskeletal network is the major determinant of axonal NF content, enabling the generation of the striking local variations in NF number seen along axons.