RESUMO
Oligodendrocytes myelinate axons for rapid impulse conduction and contribute to normal axonal functions in the central nervous system. In multiple sclerosis, demyelination is caused by autoimmune attacks, but the role of oligodendroglial cells in disease progression and axon degeneration is unclear. Here we show that oligodendrocytes harbor peroxisomes whose function is essential for maintaining white matter tracts throughout adult life. By selectively inactivating the import factor PEX5 in myelinating glia, we generated mutant mice that developed normally, but within several months showed ataxia, tremor and premature death. Absence of functional peroxisomes from oligodendrocytes caused widespread axonal degeneration and progressive subcortical demyelination, but did not interfere with glial survival. Moreover, it caused a strong proinflammatory milieu and, unexpectedly, the infiltration of B and activated CD8+ T cells into brain lesions. We conclude that peroxisomes provide oligodendrocytes with an essential neuroprotective function against axon degeneration and neuroinflammation, which is relevant for human demyelinating diseases.
Assuntos
Axônios/fisiologia , Doenças Desmielinizantes/fisiopatologia , Oligodendroglia/fisiologia , Peroxissomos/fisiologia , Animais , Axônios/patologia , Linfócitos T CD8-Positivos/imunologia , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/patologia , Humanos , Metabolismo dos Lipídeos , Camundongos , Fibras Nervosas Mielinizadas , Receptor 1 de Sinal de Orientação para Peroxissomos , Receptores Citoplasmáticos e Nucleares/fisiologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Myelination of axons by oligodendrocytes enables rapid impulse propagation in the central nervous system. But long-term interactions between axons and their myelin sheaths are poorly understood. Here we show that Cnp1, which encodes 2',3'-cyclic nucleotide phosphodiesterase in oligodendrocytes, is essential for axonal survival but not for myelin assembly. In the absence of glial cyclic nucleotide phosphodiesterase, mice developed axonal swellings and neurodegeneration throughout the brain, leading to hydrocephalus and premature death. But, in contrast to previously studied myelin mutants, the ultrastructure, periodicity and physical stability of myelin were not altered in these mice. Genetically, the chief function of glia in supporting axonal integrity can thus be completely uncoupled from its function in maintaining compact myelin. Oligodendrocyte dysfunction, such as that in multiple sclerosis lesions, may suffice to cause secondary axonal loss.
Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/fisiologia , Axônios/fisiologia , Bainha de Mielina/fisiologia , Oligodendroglia/fisiologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/deficiência , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , Animais , Axônios/patologia , Citoesqueleto/fisiologia , Feminino , Marcação de Genes , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/fisiopatologia , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , FenótipoRESUMO
Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated ax(J) gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. Ax(J) mouse mutants, characterized by cerebellar ataxia, display both increased GABA(A) receptor (GABA(A)R) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABA(A)R alpha1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA(A)R turnover at cerebellar synapses contributes to ax(J)-mediated behavioural impairment.
Assuntos
Ataxia/genética , Ataxia/metabolismo , Mutação , Receptores de GABA-A/metabolismo , Ubiquitina Tiolesterase/genética , Animais , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Ligação Proteica , Células de Purkinje/metabolismo , Receptores de GABA-A/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.
Assuntos
Microtúbulos/metabolismo , Sinapses/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Células Cultivadas , Cinesinas/metabolismo , Proteínas de Membrana/metabolismo , Ácido Poliglutâmico/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
In the developing nervous system, constitutive activation of the AKT/mTOR (mammalian target of rapamycin) pathway in myelinating glial cells is associated with hypermyelination of the brain, but is reportedly insufficient to drive myelination by Schwann cells. We have hypothesized that it requires additional mechanisms downstream of NRG1/ErbB signaling to trigger myelination in the peripheral nervous system. Here, we demonstrate that elevated levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) have developmental effects on both oligodendrocytes and Schwann cells. By generating conditional mouse mutants, we found that Pten-deficient Schwann cells are enhanced in number and can sort and myelinate axons with calibers well below 1 microm. Unexpectedly, mutant glial cells also spirally enwrap C-fiber axons within Remak bundles and even collagen fibrils, which lack any membrane surface. Importantly, PIP3-dependent hypermyelination of central axons, which is observed when targeting Pten in oligodendrocytes, can also be induced after tamoxifen-mediated Cre recombination in adult mice. We conclude that it requires distinct PIP3 effector mechanisms to trigger axonal wrapping. That myelin synthesis is not restricted to early development but can occur later in life is relevant to developmental disorders and myelin disease.
Assuntos
Bainha de Mielina/fisiologia , Oligodendroglia/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Células de Schwann/fisiologia , Envelhecimento , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Contagem de Células , Colágeno/metabolismo , Camundongos , Camundongos Transgênicos , Bainha de Mielina/ultraestrutura , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Mielinizadas/ultraestrutura , Neuroglia/fisiologia , Neuroglia/ultraestrutura , Oligodendroglia/ultraestrutura , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Células de Schwann/ultraestrutura , Nervo Isquiático/fisiologia , Nervo Isquiático/ultraestruturaRESUMO
Neuroligins are postsynaptic cell adhesion molecules that associate with presynaptic neurexins. Both factors form a transsynaptic connection, mediate signaling across the synapse, specify synaptic functions, and play a role in synapse formation. Neuroligin dysfunction impairs synaptic transmission, disrupts neuronal networks, and is thought to participate in cognitive diseases. Here we report that chemical treatment designed to induce long-term potentiation or long-term depression (LTD) induces neuroligin 1/3 turnover, leading to either increased or decreased surface membrane protein levels, respectively. Despite its structural role at a crucial transsynaptic position, GFP-neuroligin 1 leaves synapses in hippocampal neurons over time with chemical LTD-induced neuroligin internalization depending on an intact microtubule cytoskeleton. Accordingly, neuroligin 1 and its binding partner postsynaptic density protein-95 (PSD-95) associate with components of the dynein motor complex and undergo retrograde cotransport with a dynein subunit. Transgenic depletion of dynein function in mice causes postsynaptic NLG1/3 and PSD-95 enrichment. In parallel, PSD lengths and spine head sizes are significantly increased, a phenotype similar to that observed upon transgenic overexpression of NLG1 (Dahlhaus et al., 2010). Moreover, application of a competitive PSD-95 peptide and neuroligin 1 C-terminal mutagenesis each specifically alter neuroligin 1 surface membrane expression and interfere with its internalization. Our data suggest the concept that synaptic plasticity regulates neuroligin turnover through active cytoskeleton transport.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Biotinilação , Células Cultivadas , Citoesqueleto/metabolismo , Proteína 4 Homóloga a Disks-Large , Dineínas/metabolismo , Eletrofisiologia , Guanilato Quinases , Hipocampo/citologia , Imuno-Histoquímica , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , TransfecçãoRESUMO
The dynamics of postsynaptic receptor scaffold formation and remodeling at inhibitory synapses remain largely unknown. Gephyrin, which is a multimeric scaffold protein, interacts with cytoskeletal elements and stabilizes glycine receptors (GlyRs) and individual subtypes of gamma-aminobutyric acid A receptors at inhibitory postsynaptic sites. We report intracellular mobility of gephyrin transports packets over time. Gephyrin units enter and exit active synapses within several minutes. In addition to previous reports of GlyR-gephyrin interactions at plasma membranes, we show cosedimentation and coimmunoprecipitation of both proteins from vesicular fractions. Moreover, GlyR and gephyrin are cotransported within neuronal dendrites and further coimmunoprecipitate and colocalize with the dynein motor complex. As a result, the blockade of dynein function or dynein-gephyrin interaction, as well as the depolymerization of microtubules, interferes with retrograde gephyrin recruitment. Our data suggest a GlyR-gephyrin-dynein transport complex and support the concept that gephyrin-motor interactions contribute to the dynamic and activity-dependent rearrangement of postsynaptic GlyRs, a process thought to underlie the regulation of synaptic strength.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Receptores de Glicina/metabolismo , Animais , Bicuculina/farmacologia , Proteínas de Transporte/genética , Células Cultivadas , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Complexo Dinactina , Dineínas/metabolismo , Hipocampo/citologia , Humanos , Cinética , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Centro Organizador dos Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Mutação/genética , Mutação/fisiologia , Neuritos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Nocodazol/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Cloreto de Potássio/farmacologia , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Ratos , Estricnina/farmacologia , Sinaptofisina/análise , Transfecção , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/análiseRESUMO
We have selectively inhibited Notch1 signaling in oligodendrocyte precursors (OPCs) using the Cre/loxP system in transgenic mice to investigate the role of Notch1 in oligodendrocyte (OL) development and differentiation. Early development of OPCs appeared normal in the spinal cord. However, at embryonic day 17.5, premature OL differentiation was observed and ectopic immature OLs were present in the gray matter. At birth, OL apoptosis was strongly increased in Notch1 mutant animals. Premature OL differentiation was also observed in the cerebrum, indicating that Notch1 is required for the correct spatial and temporal regulation of OL differentiation in various regions of the central nervous system. These findings establish a widespread function of Notch1 in the late steps of mammalian OPC development in vivo.
Assuntos
Apoptose/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Membrana/fisiologia , Oligodendroglia/fisiologia , Receptores de Superfície Celular , Medula Espinal/fisiologia , Fatores de Transcrição , Animais , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mutação , Prosencéfalo/fisiologia , Receptor Notch1 , Medula Espinal/citologiaRESUMO
Cholesterol in the mammalian brain is a risk factor for certain neurodegenerative diseases, raising the question of its normal function. In the mature brain, the highest cholesterol content is found in myelin. We therefore created mice that lack the ability to synthesize cholesterol in myelin-forming oligodendrocytes. Mutant oligodendrocytes survived, but CNS myelination was severely perturbed, and mutant mice showed ataxia and tremor. CNS myelination continued at a reduced rate for many months, and during this period, the cholesterol-deficient oligodendrocytes actively enriched cholesterol and assembled myelin with >70% of the cholesterol content of wild-type myelin. This shows that cholesterol is an indispensable component of myelin membranes and that cholesterol availability in oligodendrocytes is a rate-limiting factor for brain maturation.
Assuntos
Colesterol/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Apolipoproteínas E/metabolismo , Comportamento Animal , Northern Blotting/métodos , Southern Blotting/métodos , Western Blotting/métodos , Membrana Celular/metabolismo , Sistema Nervoso Central/metabolismo , Colesterol/deficiência , Cromatografia em Camada Fina/métodos , Clonagem Molecular , Creatina/metabolismo , Farnesil-Difosfato Farnesiltransferase/deficiência , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Hibridização In Situ/métodos , Metabolismo dos Lipídeos , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes/fisiologia , Microscopia Eletrônica de Transmissão/métodos , Microssomos/metabolismo , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Oligodendroglia/ultraestrutura , Fenótipo , Desempenho Psicomotor/fisiologia , RNA/análise , Receptores de LDL/metabolismo , Coloração pela Prata/métodos , Medula Espinal/metabolismo , Medula Espinal/ultraestruturaRESUMO
Functional studies in neurons often require controllable simultaneous delivery of different molecules to individual cells within networks. Microinjection represents a suitable and alternative method to deliver cDNAs, oligonucleotides, siRNAs, peptides or antibodies for expression, expression knockdown or loss-of-function studies, respectively. Moreover, molecules can be systematically applied to individual neurons in a controlled manner without affecting neighbouring cells. Establishment of microinjection is often complicated and time consuming. Here we describe a simple and reliable protocol for molecular cell biologists to establish injection of various molecules (ng to microg range) to living neurons in a reasonable period of time.
Assuntos
Anticorpos/administração & dosagem , Hipocampo/citologia , Microinjeções/métodos , Neurônios/efeitos dos fármacos , Ácidos Nucleicos/administração & dosagem , Peptídeos/administração & dosagem , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Camundongos , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Abnormal cell loss is the common cause of a large number of developmental and degenerative diseases. To model such diseases in transgenic animals, we have developed a line of mice that allows the efficient depletion of virtually any cell type in vivo following somatic Cre-mediated gene recombination. By introducing the diphtheria toxin fragment A (DT-A) gene as a conditional expression construct (floxed lacZ-DT-A) into the ubiquitously expressed ROSA26 locus, we produced a line of mice that would permit cell-specific activation of the toxin gene. Following Cre-mediated recombination under the control of cell-type-specific promoters, lacZ gene expression was efficiently replaced by de novo transcription of the Cre-recombined DT-A gene. We provide proof of this principle, initially for cells of the central nervous system (pyramidal neurons and oligodendrocytes), the immune system (B cells), and liver tissue (hepatocytes), that the conditional expression of DT-A is functional in vivo, resulting in the generation of novel degenerative disease models.
Assuntos
Morte Celular/fisiologia , Toxina Diftérica/metabolismo , Integrases/metabolismo , Fragmentos de Peptídeos/metabolismo , Recombinação Genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/fisiologia , Embrião de Mamíferos/fisiologia , Regulação da Expressão Gênica , Genes Reporter , Camundongos , Camundongos Transgênicos , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The kinesin KIF21B is implicated in several human neurological disorders, including delayed cognitive development, yet it remains unclear how KIF21B dysfunction may contribute to pathology. One limitation is that relatively little is known about KIF21B-mediated physiological functions. Here, we generated Kif21b knockout mice and used cellular assays to investigate the relevance of KIF21B in neuronal and in vivo function. We show that KIF21B is a processive motor protein and identify an additional role for KIF21B in regulating microtubule dynamics. In neurons lacking KIF21B, microtubules grow more slowly and persistently, leading to tighter packing in dendrites. KIF21B-deficient neurons exhibit decreased dendritic arbor complexity and reduced spine density, which correlate with deficits in synaptic transmission. Consistent with these observations, Kif21b-null mice exhibit behavioral changes involving learning and memory deficits. Our study provides insight into the cellular function of KIF21B and the basis for cognitive decline resulting from KIF21B dysregulation.
Assuntos
Forma Celular , Cinesinas/metabolismo , Memória/fisiologia , Microtúbulos/metabolismo , Neurônios/citologia , Sinapses/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Marcação de Genes , Células HeLa , Humanos , Cinesinas/deficiência , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Camundongos Knockout , Microtúbulos/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Reprodutibilidade dos TestesRESUMO
Axoglial interactions underlie the clustering of ion channels and of cell adhesion molecules, regulate gene expression, and control cell survival. We report that Cnp1-null mice, lacking expression of the myelin protein cyclic nucleotide phosphodiesterase (CNP), have disrupted axoglial interactions in the central nervous system (CNS). Nodal sodium channels (Nav) and paranodal adhesion proteins (Caspr) are initially clustered normally, but become progressively disorganized with age. These changes are characterized by mislocalized Caspr immunostaining, combined with a decrease of clustered Na+ channels, and occur before axonal degeneration and microglial invasion, both prominent in older Cnp1-null mice. We suggest that CNP is a glial protein required for maintaining the integrity of paranodes and that disrupted axoglial signaling at this site underlies progressive axonal degeneration, observed later in the CNS of Cnp1-null mice.