RESUMO
The Neurofascins (NFASCs) are a family of proteins encoded by alternative transcripts of NFASC that cooperate in the assembly of the node of Ranvier in myelinated nerves. Differential expression of NFASC in neurons and glia presents a remarkable example of cell-type specific expression of protein isoforms with a common overall function. In mice there are three NFASC isoforms: Nfasc186 and Nfasc140, located in the axonal membrane at the node of Ranvier, and Nfasc155, a glial component of the paranodal axoglial junction. Nfasc186 and Nfasc155 are the major isoforms at mature nodes and paranodes, respectively. Conditional deletion of the glial isoform Nfasc155 in mice causes severe motor coordination defects and death at 16-17 days after birth. We describe a proband with severe congenital hypotonia, contractures of fingers and toes, and no reaction to touch or pain. Whole exome sequencing revealed a homozygous NFASC variant chr1:204953187-C>T (rs755160624). The variant creates a premature stop codon in 3 out of four NFASC human transcripts and is predicted to specifically eliminate Nfasc155 leaving neuronal Neurofascin intact. The selective absence of Nfasc155 and disruption of the paranodal junction was confirmed by an immunofluorescent study of skin biopsies from the patient versus control. We propose that the disease in our proband is the first reported example of genetic deficiency of glial Neurofascin isoforms in humans and that the severity of the condition reflects the importance of the Nfasc155 in forming paranodal axoglial junctions and in determining the structure and function of the node of Ranvier.
Assuntos
Moléculas de Adesão Celular/genética , Junções Intercelulares/metabolismo , Hipotonia Muscular/genética , Mutação , Fatores de Crescimento Neural/genética , Doenças do Sistema Nervoso/genética , Neuroglia/metabolismo , Animais , Condicionamento Psicológico , Análise Mutacional de DNA , Feminino , Homozigoto , Humanos , Lactente , Junções Intercelulares/genética , Camundongos , Hipotonia Muscular/metabolismo , Doenças do Sistema Nervoso/metabolismo , Polônia , Isoformas de Proteínas , SíndromeRESUMO
Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS) and central nervous system (CNS) myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK)-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1), a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K)/v-AKT murine thymoma viral oncogene homolog (AKT) pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS.
Assuntos
Sistema Nervoso Central/fisiologia , Cinesinas/fisiologia , Proteínas de Membrana/fisiologia , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sistema Nervoso Periférico/fisiologia , Animais , Proteína 1 Homóloga a Discs-Large , Camundongos , Camundongos Knockout , Oligodendroglia/metabolismo , Proteínas Associadas SAP90-PSD95 , Células de Schwann/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, primarily affecting lower motor neurons. Recent evidence from SMA and related conditions suggests that glial cells can influence disease severity. Here, we investigated the role of glial cells in the peripheral nervous system by creating SMA mice selectively overexpressing SMN in myelinating Schwann cells (Smn-/-;SMN2tg/0;SMN1SC). Restoration of SMN protein levels restricted solely to Schwann cells reversed myelination defects, significantly improved neuromuscular function and ameliorated neuromuscular junction pathology in SMA mice. However, restoration of SMN in Schwann cells had no impact on motor neuron soma loss from the spinal cord or ongoing systemic and peripheral pathology. This study provides evidence for a defined, intrinsic contribution of glial cells to SMA disease pathogenesis and suggests that therapies designed to include Schwann cells in their target tissues are likely to be required in order to rescue myelination defects and associated disease symptoms.
Assuntos
Neuroglia/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/metabolismo , Bainha de Mielina/metabolismo , Degeneração Neural/patologia , Doenças Neuromusculares/patologia , Junção Neuromuscular/metabolismo , Células de Schwann/metabolismo , Medula Espinal/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
UNLABELLED: Schwann cells (SCs), ensheathing glia of the peripheral nervous system, support axonal survival and function. Abnormalities in SC metabolism affect their ability to provide this support and maintain axon integrity. To further interrogate this metabolic influence on axon-glial interactions, we generated OGT-SCKO mice with SC-specific deletion of the metabolic/nutrient sensing protein O-GlcNAc transferase that mediates the O-linked addition of N-acetylglucosamine (GlcNAc) moieties to Ser and Thr residues. The OGT-SCKO mice develop tomaculous demyelinating neuropathy characterized by focal thickenings of the myelin sheath (tomacula), progressive demyelination, axonal loss, and motor and sensory nerve dysfunction. Proteomic analysis identified more than 100 O-GlcNAcylated proteins in rat sciatic nerve, including Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans. PRX lacking O-GlcNAcylation is mislocalized within the myelin sheath of these mutant animals. Furthermore, phenotypes of OGT-SCKO and Prx-deficient mice are very similar, suggesting that metabolic control of PRX O-GlcNAcylation is crucial for myelin maintenance and axonal integrity. SIGNIFICANCE STATEMENT: The nutrient sensing protein O-GlcNAc transferase (OGT) mediates post-translational O-linked N-acetylglucosamine (GlcNAc) modification. Here we find that OGT functions in Schwann cells (SCs) to maintain normal myelin and prevent axonal loss. SC-specific deletion of OGT (OGT-SCKO mice) causes a tomaculous demyelinating neuropathy accompanied with progressive axon degeneration and motor and sensory nerve dysfunction. We also found Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans, is O-GlcNAcylated. Importantly, phenotypes of OGT-SCKO and Prx mutant mice are very similar, implying that compromised PRX function contributes to the neuropathy of OGT-SCKO mice. This study will be useful in understanding how SC metabolism contributes to PNS function and in developing new strategies for treating peripheral neuropathy by targeting SC function.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/patologia , Proteínas de Membrana/metabolismo , Bainha de Mielina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Nervo Isquiático/metabolismo , Acetilglucosamina/metabolismo , Potenciais de Ação/genética , Animais , Doenças Autoimunes do Sistema Nervoso/fisiopatologia , Axônios/patologia , Axônios/ultraestrutura , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Glucose/metabolismo , Glicosilação , Humanos , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/fisiologia , Bainha de Mielina/ultraestrutura , N-Acetilglucosaminiltransferases/genética , Proteínas do Tecido Nervoso/metabolismo , Condução Nervosa/genética , Proteômica , Nervo Isquiático/patologia , Nervo Isquiático/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: All-oral combination therapy is desirable for patients with chronic hepatitis C virus (HCV) infection. We evaluated daclatasvir (an HCV NS5A replication complex inhibitor) plus sofosbuvir (a nucleotide analogue HCV NS5B polymerase inhibitor) in patients infected with HCV genotype 1, 2, or 3. METHODS: In this open-label study, we initially randomly assigned 44 previously untreated patients with HCV genotype 1 infection and 44 patients infected with HCV genotype 2 or 3 to daclatasvir at a dose of 60 mg orally once daily plus sofosbuvir at a dose of 400 mg orally once daily, with or without ribavirin, for 24 weeks. The study was expanded to include 123 additional patients with genotype 1 infection who were randomly assigned to daclatasvir plus sofosbuvir, with or without ribavirin, for 12 weeks (82 previously untreated patients) or 24 weeks (41 patients who had previous virologic failure with telaprevir or boceprevir plus peginterferon alfa-ribavirin). The primary end point was a sustained virologic response (an HCV RNA level of <25 IU per milliliter) at week 12 after the end of therapy. RESULTS: Overall, 211 patients received treatment. Among patients with genotype 1 infection, 98% of 126 previously untreated patients and 98% of 41 patients who did not have a sustained virologic response with HCV protease inhibitors had a sustained virologic response at week 12 after the end of therapy. A total of 92% of 26 patients with genotype 2 infection and 89% of 18 patients with genotype 3 infection had a sustained virologic response at week 12. High rates of sustained virologic response at week 12 were observed among patients with HCV subtypes 1a and 1b (98% and 100%, respectively) and those with CC and non-CC IL28B genotypes (93% and 98%, respectively), as well as among patients who received ribavirin and those who did not (94% and 98%, respectively). The most common adverse events were fatigue, headache, and nausea. CONCLUSIONS: Once-daily oral daclatasvir plus sofosbuvir was associated with high rates of sustained virologic response among patients infected with HCV genotype 1, 2, or 3, including patients with no response to prior therapy with telaprevir or boceprevir. (Funded by Bristol-Myers Squibb and Pharmasset (Gilead); A1444040 ClinicalTrials.gov number, NCT01359644.).
Assuntos
Antivirais/uso terapêutico , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Imidazóis/uso terapêutico , Uridina Monofosfato/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Adulto , Idoso , Antivirais/efeitos adversos , Carbamatos , Quimioterapia Combinada , Feminino , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/isolamento & purificação , Humanos , Imidazóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteases/uso terapêutico , Pirrolidinas , RNA Viral/análise , Ribavirina/uso terapêutico , Sofosbuvir , Uridina Monofosfato/efeitos adversos , Uridina Monofosfato/uso terapêutico , Valina/análogos & derivados , Adulto JovemRESUMO
Rapid nerve conduction in myelinated nerves requires the clustering of voltage-gated sodium channels at nodes of Ranvier. The Neurofascin (Nfasc) gene has a unique role in node formation because it encodes glial and neuronal isoforms of neurofascin (Nfasc155 and Nfasc186, respectively) with key functions in assembling the nodal macromolecular complex. A third neurofascin, Nfasc140, has also been described; however, neither the cellular origin nor function of this isoform was known. Here we show that Nfasc140 is a neuronal protein strongly expressed during mouse embryonic development. Expression of Nfasc140 persists but declines during the initial stages of node formation, in contrast to Nfasc155 and Nfasc186, which increase. Nevertheless, Nfasc140, like Nfasc186, can cluster voltage-gated sodium channels (Nav) at the developing node of Ranvier and can restore electrophysiological function independently of Nfasc155 and Nfasc186. This suggests that Nfasc140 complements the function of Nfasc155 and Nfasc186 in initial stages of the assembly and stabilization of the nodal complex. Further, Nfasc140 is reexpressed in demyelinated white matter lesions of postmortem brain tissue from human subjects with multiple sclerosis. This expands the critical role of the Nfasc gene in the function of myelinated axons and reveals further redundancy in the mechanisms required for the formation of this crucial structure in the vertebrate nervous system.
Assuntos
Moléculas de Adesão Celular/metabolismo , Fatores de Crescimento Neural/metabolismo , Nós Neurofibrosos/metabolismo , Rombencéfalo/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Axônios/metabolismo , Estudos de Casos e Controles , Moléculas de Adesão Celular/genética , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Fatores de Crescimento Neural/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Rombencéfalo/embriologia , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Fast, saltatory conduction in myelinated nerves requires the clustering of voltage-gated sodium channels (Nav) at nodes of Ranvier in a nodal complex. The Neurofascin (Nfasc) gene encodes neuronal Neurofascin 186 (Nfasc186) at the node and glial Neurofascin 155 at the paranode, and these proteins play a key role in node assembly. However, their role in the maintenance and stability of the node is less well understood. Here we show that by inducible ablation of Nfasc in neurons in adult mice, Nfasc186 expression is reduced by >99% and 94% at PNS and CNS nodes, respectively. Gliomedin and NrCAM at PNS and brevican at CNS nodes are largely lost with neuronal neurofascin; however, Nav at nodes of Ranvier persist, albeit with â¼40% reduction in expression levels. ßIV Spectrin, ankyrin G, and, to a lesser extent, the ß1 subunit of the sodium channel, are less affected at the PNS node than in the CNS. Nevertheless, there is a 38% reduction in PNS conduction velocity. Loss of Nfasc186 provokes CNS paranodal disorganization, but this does not contribute to loss of Nav. These results show that Nav at PNS nodes are still maintained in a nodal complex when neuronal neurofascin is depleted, whereas the retention of nodal Nav in the CNS, despite more extensive dissolution of the complex, suggests a supportive role for the partially disrupted paranodal axoglial junction in selectively maintaining Nav at the CNS node.
Assuntos
Moléculas de Adesão Celular/genética , Deleção de Genes , Fatores de Crescimento Neural/genética , Nós Neurofibrosos/metabolismo , Medula Espinal/metabolismo , Animais , Brevicam/metabolismo , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Feminino , Masculino , Camundongos , Fatores de Crescimento Neural/metabolismo , Neuroglia/metabolismo , Transporte Proteico , Medula Espinal/citologia , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Postnatal synapse elimination plays a critical role in sculpting and refining neural connectivity throughout the central and peripheral nervous systems, including the removal of supernumerary axonal inputs from neuromuscular junctions (NMJs). Here, we reveal a novel and important role for myelinating glia in regulating synapse elimination at the mouse NMJ, where loss of a single glial cell protein, the glial isoform of neurofascin (Nfasc155), was sufficient to disrupt postnatal remodeling of synaptic circuitry. Neuromuscular synapses were formed normally in mice lacking Nfasc155, including the establishment of robust neuromuscular synaptic transmission. However, loss of Nfasc155 was sufficient to cause a robust delay in postnatal synapse elimination at the NMJ across all muscle groups examined. Nfasc155 regulated neuronal remodeling independently of its canonical role in forming paranodal axo-glial junctions, as synapse elimination occurred normally in mice lacking the axonal paranodal protein Caspr. Rather, high-resolution proteomic screens revealed that loss of Nfasc155 from glial cells was sufficient to disrupt neuronal cytoskeletal organization and trafficking pathways, resulting in reduced levels of neurofilament light (NF-L) protein in distal axons and motor nerve terminals. Mice lacking NF-L recapitulated the delayed synapse elimination phenotype observed in mice lacking Nfasc155, suggesting that glial cells regulate synapse elimination, at least in part, through modulation of the axonal cytoskeleton. Together, our study reveals a glial cell-dependent pathway regulating the sculpting of neuronal connectivity and synaptic circuitry in the peripheral nervous system.
Assuntos
Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/fisiologia , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/fisiologia , Junção Neuromuscular/fisiologia , Sinapses/fisiologia , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/fisiologia , Citoesqueleto/metabolismo , Camundongos , Camundongos Knockout , Placa Motora/crescimento & desenvolvimento , Neurônios Motores/metabolismo , Fatores de Crescimento Neural/genética , Condução Nervosa/genética , Condução Nervosa/fisiologia , Proteínas de Neurofilamentos/metabolismo , Neuroglia/metabolismo , Junção Neuromuscular/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Proteômica , Células de Schwann/metabolismo , Sinapses/genética , Transmissão Sináptica/fisiologiaRESUMO
The influences of axon diameter, myelin thickness, and internodal length on the velocity of conduction of peripheral nerve action potentials are unclear. Previous studies have demonstrated a strong dependence of conduction velocity on internodal length. However, a theoretical analysis has suggested that this relationship may be lost above a nodal separation of â¼0.6 mm. Here we measured nerve conduction velocities in a rabbit model of limb lengthening that produced compensatory increases in peripheral nerve growth. Divided tibial bones in one hindlimb were gradually lengthened at 0.7 mm per day using an external frame attached to the bone. This was associated with a significant increase (33%) of internodal length (0.95-1.3 mm) in axons of the tibial nerve that varied in proportion to the mechanical strain in the nerve of the lengthened limb. Axonal diameter, myelin thickness, and g-ratios were not significantly altered by limb lengthening. Despite the substantial increase in internodal length, no significant change was detected in conduction velocity (â¼43 m/s) measured either in vivo or in isolated tibial nerves. The results demonstrate that the internode remains plastic in the adult but that increases in internodal length of myelinated adult nerve axons do not result in either deficiency or proportionate increases in their conduction velocity and support the view that the internodal lengths of nerves reach a plateau beyond which their conduction velocities are no longer sensitive to increases in internodal length.
Assuntos
Potenciais de Ação/fisiologia , Membro Posterior/fisiologia , Condução Nervosa/fisiologia , Nós Neurofibrosos/fisiologia , Nervo Tibial/fisiologia , Animais , Estimulação Elétrica , Membro Posterior/inervação , Técnicas In Vitro , Masculino , Microscopia Eletrônica de Transmissão , Fibras Nervosas Mielinizadas/fisiologia , Coelhos , Nós Neurofibrosos/ultraestrutura , Tempo de Reação/fisiologia , Nervo Tibial/ultraestruturaRESUMO
BMS-791325 is a nonnucleoside inhibitor of hepatitis C virus (HCV) NS5B polymerase with low-nanomolar potency against genotypes 1a (50% effective concentration [EC50], 3 nM) and 1b (EC50, 7 nM) in vitro. BMS-791325 safety, pharmacokinetics, and antiviral activity were evaluated in a double-blind, placebo-controlled, single-ascending-dose study in 24 patients (interferon naive and experienced) with chronic HCV genotype 1 infection, randomized (5:1) to receive a single dose of BMS-791325 (100, 300, 600, or 900 mg) or placebo. The prevalence and phenotype of HCV variants at baseline and specific posttreatment time points were assessed. Antiviral activity was observed in all cohorts, with a mean HCV RNA decline of ≈2.5 log10 copies/ml observed 24 h after a single 300-mg dose. Mean plasma half-life among cohorts was 7 to 9 h; individual 24-hour levels exceeded the protein-adjusted EC90 for genotype 1 at all doses. BMS-791325 was generally well tolerated, with no serious adverse events or discontinuations. Enrichment for resistance variants was not observed at 100 to 600 mg. At 900 mg, variants (P495L/S) associated with BMS-791325 resistance in vitro were transiently observed in one patient, concurrent with an observed HCV RNA decline of 3.4 log10 IU/ml, but were replaced with wild type by 48 h. Single doses of BMS-791325 were well tolerated; demonstrated rapid, substantial, and exposure-related antiviral activity; displayed dose-related increases in exposure; and showed viral kinetic and pharmacokinetic profiles supportive of once- or twice-daily dosing. These results support its further development in combination with other direct-acting antivirals for HCV genotype 1 infection. (This trial has been registered at ClinicalTrials.gov under registration no. NCT00664625.).
Assuntos
Antivirais/farmacocinética , Benzazepinas/farmacocinética , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Indóis/farmacocinética , Proteínas não Estruturais Virais/antagonistas & inibidores , Adulto , Antivirais/administração & dosagem , Antivirais/sangue , Antivirais/química , Benzazepinas/administração & dosagem , Benzazepinas/sangue , Benzazepinas/química , Estudos de Coortes , Método Duplo-Cego , Farmacorresistência Viral , Feminino , Genótipo , Meia-Vida , Hepacivirus/classificação , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Humanos , Indóis/administração & dosagem , Indóis/sangue , Indóis/química , Interferons , Masculino , Pessoa de Meia-Idade , Fenótipo , RNA Viral/sangue , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Adulto JovemRESUMO
Cajal bands are cytoplasmic channels flanked by appositions where the abaxonal surface of Schwann cell myelin apposes and adheres to the overlying plasma membrane. These appositions contain a dystroglycan complex that includes periaxin and dystrophin-related protein 2 (Drp2). Loss of periaxin disrupts appositions and Cajal bands in Schwann cells and causes a severe demyelinating neuropathy in mouse and human. Here, we investigated the role of mouse Drp2 in apposition assembly and Cajal band function and compared it with periaxin. We show that periaxin and Drp2 are not only both required to form appositions, but they must also interact. Periaxin-Drp2 interaction is also required for Drp2 phosphorylation, but phosphorylation is not required for the assembly of appositions. Drp2 loss causes corresponding increases in Dystrophin family members, utrophin and dystrophin Dp116, although dystroglycan remains unchanged. We also show that all dystroglycan complexes in Schwann cells use the uncleaved form of ß-dystroglycan. Drp2-null Schwann cells have disrupted appositions and Cajal bands, and they undergo focal hypermyelination and concomitant demyelination. Nevertheless, they do not have the short internodal lengths and associated reduced nerve conduction velocity seen in the absence of periaxin, showing that periaxin regulates Schwann cell elongation independent of its role in the dystroglycan complex. We conclude that the primary role of the dystroglycan complex in appositions is to stabilize and limit the radial growth of myelin.
Assuntos
Distroglicanas/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células de Schwann/fisiologia , Animais , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Compressão Nervosa/métodos , Proteínas do Tecido Nervoso/genética , Células de Schwann/citologia , Neuropatia Ciática/metabolismo , Neuropatia Ciática/patologia , Neuropatia Ciática/fisiopatologiaRESUMO
In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell.
Assuntos
Axônios/patologia , Bainha de Mielina/patologia , Células de Schwann/patologia , Serina-Treonina Quinases TOR/deficiência , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Crescimento Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/genética , Bainha de Mielina/ultraestrutura , Técnicas de Cultura de Órgãos , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura , Serina-Treonina Quinases TOR/genéticaRESUMO
Transparency of the ocular lens depends on symmetric packing and membrane organization of highly elongated hexagonal fiber cells. These cells possess an extensive, well-ordered cortical cytoskeleton to maintain cell shape and to anchor membrane components. Periaxin (Prx), a PDZ domain protein involved in myelin sheath stabilization, is also a component of adhaerens plaques in lens fiber cells. Here we show that Prx is expressed in lens fibers and exhibits maturation dependent redistribution, clustering discretely at the tricellular junctions in mature fiber cells. Prx exists in a macromolecular complex with proteins involved in membrane organization including ankyrin-B, spectrin, NrCAM, filensin, ezrin and desmoyokin. Importantly, Prx knockout mouse lenses were found to be softer and more easily deformed than normal lenses, revealing disruptions in fiber cell hexagonal packing, membrane skeleton and membrane stability. These observations suggest a key role for Prx in maturation, packing, and membrane organization of lens fiber cells. Hence, there may be functional parallels between the roles of Prx in membrane stabilization of the myelin sheath and the lens fiber cell.
Assuntos
Membrana Celular/ultraestrutura , Cristalino/citologia , Proteínas de Membrana/fisiologia , Animais , Membrana Celular/metabolismo , Forma Celular , Imunofluorescência , Cristalino/embriologia , Cristalino/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica de TransmissãoRESUMO
Syncoilin is an atypical type III intermediate filament (IF) protein, which is expressed in muscle and is associated with the dystrophin-associated protein complex. Here, we show that syncoilin is expressed in both the central and peripheral nervous systems. Isoform Sync1 is dominant in the brain, but isoform Sync2 is dominant in the spinal cord and sciatic nerve. Peripherin is a type III IF protein that has been shown to colocalise and interact with syncoilin. Our analyses suggest that syncoilin might function to modulate formation of peripherin filament networks through binding to peripherin isoforms. Peripherin is associated with the disease amyotrophic lateral sclerosis (ALS), thus establishing a link between syncoilin and ALS. A neuronal analysis of the syncoilin-null mouse (Sync(-/-)) revealed a reduced ability in accelerating treadmill and rotarod tests. This phenotype might be attributable to the impaired function of extensor digitorum longus muscle and type IIb fibres caused by a shift from large- to small-calibre motor axons in the ventral root.
Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Glicoproteínas de Membrana/metabolismo , Neurônios Motores/metabolismo , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/metabolismo , Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/genética , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Periferinas , Reação em Cadeia da Polimerase , Ligação Proteica , Isoformas de Proteínas/genética , Nervo Isquiático/metabolismo , Medula Espinal/metabolismoRESUMO
Signaling by laminins and axonal neuregulin has been implicated in regulating axon sorting by myelin-forming Schwann cells. However, the signal transduction mechanisms are unknown. Focal adhesion kinase (FAK) has been linked to alpha6beta1 integrin and ErbB receptor signaling, and we show that myelination by Schwann cells lacking FAK is severely impaired. Mutant Schwann cells could interdigitate between axon bundles, indicating that FAK signaling was not required for process extension. However, Schwann cell FAK was required to stimulate cell proliferation, suggesting that amyelination was caused by insufficient Schwann cells. ErbB2 receptor and AKT were robustly phosphorylated in mutant Schwann cells, indicating that neuregulin signaling from axons was unimpaired. These findings demonstrate the vital relationship between axon defasciculation and Schwann cell number and show the importance of FAK in regulating cell proliferation in the developing nervous system.
Assuntos
Axônios/enzimologia , Comunicação Celular/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Sistema Nervoso/enzimologia , Células de Schwann/metabolismo , Animais , Axônios/patologia , Axônios/ultraestrutura , Contagem de Células , Feminino , Quinase 1 de Adesão Focal/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Bainha de Mielina/enzimologia , Bainha de Mielina/patologia , Sistema Nervoso/embriologia , Sistema Nervoso/patologia , Gravidez , Células de Schwann/patologia , Células de Schwann/ultraestrutura , Transdução de Sinais/fisiologiaRESUMO
Voltage-gated sodium channels cluster in macromolecular complexes at nodes of Ranvier to promote rapid nerve impulse conduction in vertebrate nerves. Node assembly in peripheral nerves is thought to be initiated at heminodes at the extremities of myelinating Schwann cells, and fusion of heminodes results in the establishment of nodes. Here we show that assembly of 'early clusters' of nodal proteins in the murine axonal membrane precedes heminode formation. The neurofascin (Nfasc) proteins are essential for node assembly, and the formation of early clusters also requires neuronal Nfasc. Early clusters are mobile and their proteins are dynamically recruited by lateral diffusion. They can undergo fusion not only with each other but also with heminodes, thus contributing to the development of nodes in peripheral axons. The formation of early clusters constitutes the earliest stage in peripheral node assembly and expands the repertoire of strategies that have evolved to establish these essential structures.
Assuntos
Interneurônios/metabolismo , Proteína Nodal/metabolismo , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/metabolismo , Feminino , Gânglios Espinais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/metabolismo , Condução Nervosa , Sistema Nervoso Periférico , Células de Schwann/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
In the brain's gray matter, astrocytes regulate synapse properties, but their role is unclear for the white matter, where myelinated axons rapidly transmit information between gray matter areas. We found that in rodents, neuronal activity raised the intracellular calcium concentration ([Ca2+]i) in astrocyte processes located near action potentialgenerating sites in the axon initial segment (AIS) and nodes of Ranvier of myelinated axons. This released adenosine triphosphate, which was converted extracellularly to adenosine and thus, through A2a receptors, activated HCN2-containing cation channels that regulate two aspects of myelinated axon function: excitability of the AIS and speed of action potential propagation. Variations in astrocyte-derived adenosine level between wake and sleep states or during energy deprivation could thus control white matter information flow and neural circuit function.
Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/fisiologia , Axônios/fisiologia , Cálcio/fisiologia , Excitabilidade Cortical , Condução Nervosa , Potenciais de Ação , Animais , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Ratos Sprague-DawleyRESUMO
Neuronal remodeling and myelination are two fundamental processes during neurodevelopment. How they influence each other remains largely unknown, even though their coordinated execution is critical for circuit function and often disrupted in neuropsychiatric disorders. It is unclear whether myelination stabilizes axon branches during remodeling or whether ongoing remodeling delays myelination. By modulating synaptic transmission, cytoskeletal dynamics, and axonal transport in mouse motor axons, we show that local axon remodeling delays myelination onset and node formation. Conversely, glial differentiation does not determine the outcome of axon remodeling. Delayed myelination is not due to a limited supply of structural components of the axon-glial unit but rather is triggered by increased transport of signaling factors that initiate myelination, such as neuregulin. Further, transport of promyelinating signals is regulated via local cytoskeletal maturation related to activity-dependent competition. Our study reveals an axon branch-specific fine-tuning mechanism that locally coordinates axon remodeling and myelination.
Assuntos
Axônios , Neurônios Motores/metabolismo , Bainha de Mielina/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Transmissão SinápticaRESUMO
Nerve impulses are propagated at nodes of Ranvier in the myelinated nerves of vertebrates. Internodal distances have been proposed to affect the velocity of nerve impulse conduction; however, direct evidence is lacking, and the cellular mechanisms that might regulate the length of the myelinated segments are unknown. Ramón y Cajal described longitudinal and transverse bands of cytoplasm or trabeculae in internodal Schwann cells and suggested that they had a nutritive function. Here we show that internodal growth in wild-type nerves is precisely matched to nerve extension, but disruption of the cytoplasmic bands in Periaxin-null mice impairs Schwann cell elongation during nerve growth. By contrast, myelination proceeds normally. The capacity of wild-type and mutant Schwann cells to elongate is cell-autonomous, indicating that passive stretching can account for the lengthening of the internode during limb growth. As predicted on theoretical grounds, decreased internodal distances strikingly decrease conduction velocities and so affect motor function. We propose that microtubule-based transport in the longitudinal bands of Cajal permits internodal Schwann cells to lengthen in response to axonal growth, thus ensuring rapid nerve impulse transmission.
Assuntos
Fibras Nervosas Mielinizadas/fisiologia , Células de Schwann/citologia , Células de Schwann/fisiologia , Transmissão Sináptica/fisiologia , Animais , Axônios/fisiologia , Comportamento Animal/fisiologia , Tamanho Celular , Citoplasma/metabolismo , Deleção de Genes , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Músculo Esquelético/inervação , Proteína Básica da Mielina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nervo Isquiático/citologia , Nervo Isquiático/fisiologiaRESUMO
Ion channel complexes promote action potential initiation at the mammalian axon initial segment (AIS), and modulation of AIS size by recruitment or loss of proteins can influence neuron excitability. Although endocytosis contributes to AIS turnover, how membrane proteins traffic to this proximal axonal domain is incompletely understood. Neurofascin186 (Nfasc186) has an essential role in stabilising the AIS complex to the proximal axon, and the AIS channel protein Kv7.3 regulates neuron excitability. Therefore, we have studied how these proteins reach the AIS. Vesicles transport Nfasc186 to the soma and axon terminal where they fuse with the neuronal plasma membrane. Nfasc186 is highly mobile after insertion in the axonal membrane and diffuses bidirectionally until immobilised at the AIS through its interaction with AnkyrinG. Kv7.3 is similarly recruited to the AIS. This study reveals how key proteins are delivered to the AIS and thereby how they may contribute to its functional plasticity.