RESUMO
Neuronal-activity-dependent transcription couples sensory experience to adaptive responses of the brain including learning and memory. Mechanisms of activity-dependent gene expression including alterations of the epigenome have been characterized1-8. However, the fundamental question of whether sensory experience remodels chromatin architecture in the adult brain in vivo to induce neural code transformations and learning and memory remains to be addressed. Here we use in vivo calcium imaging, optogenetics and pharmacological approaches to show that granule neuron activation in the anterior dorsal cerebellar vermis has a crucial role in a delay tactile startle learning paradigm in mice. Of note, using large-scale transcriptome and chromatin profiling, we show that activation of the motor-learning-linked granule neuron circuit reorganizes neuronal chromatin including through long-distance enhancer-promoter and transcriptionally active compartment interactions to orchestrate distinct granule neuron gene expression modules. Conditional CRISPR knockout of the chromatin architecture regulator cohesin in anterior dorsal cerebellar vermis granule neurons in adult mice disrupts enhancer-promoter interactions, activity-dependent transcription and motor learning. These findings define how sensory experience patterns chromatin architecture and neural circuit coding in the brain to drive motor learning.
Assuntos
Retroalimentação Sensorial , Genoma , Aprendizagem/fisiologia , Destreza Motora/fisiologia , Vias Neurais , Plasticidade Neuronal/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Vermis Cerebelar/citologia , Vermis Cerebelar/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Feminino , Masculino , Camundongos , Fibras Musgosas Hipocampais , Regiões Promotoras Genéticas/genética , Células de Purkinje , Reflexo de SobressaltoRESUMO
In this Letter, '≥' should be '≤' in the sentence: "Intra-chromosomal reads were further split into short-range reads (≥1 kb) and long-range reads (>1 kb)". This error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Here we have characterized the functional impairments resulting from conditional knockout of the ubiquitin-conjugating E2 enzyme (UBC13) in rodent cerebellar granule neurons, which greatly increases the parallel fiber presynaptic boutons and functional parallel fiber/Purkinje cell synapses. We report that conditional UBC13 knockout mice exhibit reliable deficits on several gait-related variables when their velocity of ambulation is tightly controlled by a moving treadmill and by restricting space for movement. Selected gait parameters and movement patterns related to spontaneous exploration in an open field may also be affected in conditional UBC13 knockout mice. Analysis of open-field data as a function of test session half using force-plate actometer instrumentation suggest that conditional UBC13 knockout mice have alterations in emotionality, possibly affecting gait and movement variables. These findings suggest that conditional UBC13 knockout mice represent a valuable platform for assessing the effects of disturbances in cerebellar granule cell circuitry on gait and other aspects of locomotion. Also, the possibility that psychological factors such as altered emotionality may impact gait and movement patterns in these mice suggest that these mice may provide a useful model for evaluating analogous behavioral impairments in autism spectrum disorders and other neurodevelopmental syndromes associated with deregulation of ubiquitin signaling.
Assuntos
Comportamento Exploratório/fisiologia , Marcha/fisiologia , Locomoção/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Análise de Variância , Animais , Feminino , Marcha/genética , Locomoção/genética , Masculino , Camundongos , Camundongos Knockout , Enzimas de Conjugação de Ubiquitina/deficiência , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Although ubiquitin ligases have been implicated in autism, their roles and mechanisms in brain development remain incompletely understood. Here, we report that in vivo knockdown or conditional knockout of the autism-linked ubiquitin ligase RNF8 or associated ubiquitin-conjugating enzyme UBC13 in rodent cerebellar granule neurons robustly increases the number of parallel fiber presynaptic boutons and functional parallel fiber/Purkinje cell synapses. In contrast to the role of nuclear RNF8 in proliferating cells, RNF8 operates in the cytoplasm in neurons to suppress synapse differentiation in vivo. Proteomics analyses reveal that neuronal RNF8 interacts with the HECT domain protein HERC2 and scaffold protein NEURL4, and knockdown of HERC2 or NEURL4 phenocopies the inhibition of RNF8/UBC13 signaling on synapse differentiation. In behavior analyses, granule neuron-specific knockout of RNF8 or UBC13 impairs cerebellar-dependent learning. Our study defines RNF8 and UBC13 as components of a novel cytoplasmic ubiquitin-signaling network that suppresses synapse formation in the brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Neurônios/metabolismo , Sinapses/ultraestrutura , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Animais , Cerebelo/citologia , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Microscopia Eletrônica , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Proteômica , Células de Purkinje/metabolismo , Células de Purkinje/ultraestrutura , Ratos , Transdução de Sinais , Sinapses/metabolismoRESUMO
Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.
Assuntos
Hipocampo/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-B/genética , Transmissão Sináptica/genética , Animais , Giro Denteado/metabolismo , Giro Denteado/patologia , Giro Denteado/ultraestrutura , Embrião de Mamíferos , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/patologia , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/patologia , Hipocampo/ultraestrutura , Humanos , Injeções Intraventriculares , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural , Neurogênese/genética , Neurônios/patologia , Neurônios/ultraestrutura , Cultura Primária de Células , Ratos , Ratos Wistar , Receptor Cross-Talk , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Sinapses/metabolismo , Sinapses/patologia , Sinapses/ultraestruturaRESUMO
During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15-/- mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.
Assuntos
Transtornos Cognitivos/genética , Proteínas Ativadoras de GTPase/metabolismo , Hipocampo/fisiopatologia , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Comportamento Animal/fisiologia , Movimento Celular/genética , Células Cultivadas , Transtornos Cognitivos/etiologia , Feminino , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/patologia , Interneurônios/patologia , Masculino , Memória de Curto Prazo/fisiologia , Camundongos Mutantes , Neurônios/patologia , Neuropeptídeos/genética , Ratos , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Dendrites play a central role in the integration and flow of information in the nervous system. The morphogenesis and maturation of dendrites is hence an essential step in the establishment of neuronal connectivity. Recent studies have uncovered crucial functions for extrinsic cues in the development of dendrites. We review the contribution of secreted polypeptide growth factors, contact-mediated proteins, and neuronal activity in distinct phases of dendrite development. We also highlight how extrinsic cues influence local and global intracellular mechanisms of dendrite morphogenesis. Finally, we discuss how these studies have advanced our understanding of neuronal connectivity and have shed light on the pathogenesis of neurodevelopmental disorders.
Assuntos
Sinais (Psicologia) , Dendritos/fisiologia , Morfogênese/fisiologia , Neurônios/citologia , Animais , Sinalização do Cálcio , Humanos , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismoRESUMO
Intellectual disability is a prevalent disorder that remains incurable. Mutations of the X-linked protein PHF6 cause the intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS). However, the biological role of PHF6 relevant to BFLS pathogenesis has remained unknown. We report that knockdown of PHF6 profoundly impairs neuronal migration in the mouse cerebral cortex in vivo, leading to the formation of white matter heterotopias displaying neuronal hyperexcitability. We find that PHF6 physically associates with the PAF1 transcription elongation complex, and inhibition of PAF1 phenocopies the PHF6 knockdown-induced migration phenotype in vivo. We also identify Neuroglycan C/Chondroitin sulfate proteoglycan 5 (NGC/CSPG5), a potential schizophrenia susceptibility gene, as a critical downstream target of PHF6 in the control of neuronal migration. These findings define PHF6, PAF1, and NGC/CSPG5 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the brain, with important implications for the pathogenesis of developmental disorders of cognition.
Assuntos
Proteínas de Transporte/genética , Movimento Celular/fisiologia , Genes Ligados ao Cromossomo X/genética , Proteínas de Homeodomínio/genética , Deficiência Intelectual/genética , Neurônios/fisiologia , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Proteínas de Transporte/metabolismo , Feminino , Técnicas de Silenciamento de Genes/métodos , Proteínas de Homeodomínio/metabolismo , Camundongos , Gravidez , Ligação Proteica/fisiologia , Ratos , Proteínas RepressorasRESUMO
Mutations in TSPAN7--a member of the tetraspanin protein superfamily--are implicated in some forms of X-linked intellectual disability. Here we show that TSPAN7 overexpression promotes the formation of filopodia and dendritic spines in cultured hippocampal neurons from embryonic rats, whereas TSPAN7 silencing reduces head size and stability of spines and AMPA receptor currents. Via its C terminus, TSPAN7 interacts with the PDZ domain of protein interacting with C kinase 1 (PICK1), to regulate PICK1 and GluR2/3 association and AMPA receptor trafficking. These findings indicate that, in hippocampal neurons, TSPAN7 regulates AMPA receptor trafficking by limiting PICK1 accessibility to AMPA receptors and suggest an additional mechanism for the functional maturation of glutamatergic synapses, whose impairment is implicated in intellectual disability.
Assuntos
Espinhas Dendríticas/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Tetraspaninas/metabolismo , Análise de Variância , Animais , Biofísica , Proteínas de Transporte/metabolismo , Células Cultivadas , Chlorocebus aethiops , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/genética , Proteína 4 Homóloga a Disks-Large , Estimulação Elétrica , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Hidrazonas/farmacologia , Imunoprecipitação , Técnicas In Vitro , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/metabolismo , Técnicas de Patch-Clamp , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Pseudópodes/efeitos dos fármacos , Pseudópodes/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Sinapses/genética , Tetraspaninas/genética , Fatores de Tempo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Intellectual disability (ID) is a common and highly heterogeneous paediatric disorder with a very severe social impact. Intellectual disability can be caused by environmental and/or genetic factors. Although in the last two decades a number of genes have been discovered whose mutations cause mental retardation, we are still far from identifying the impact of these mutations on brain functions. Many of the genes mutated in ID code for several proteins with a variety of functions: chromatin remodelling, pre-/post-synaptic activity, and intracellular trafficking. The prevailing hypothesis suggests that the ID phenotype could emerge from abnormal cellular processing leading to pre- and/or post-synaptic dysfunction. In this chapter, we focus on the role of small GTPases and adhesion molecules, and we discuss the mechanisms through which they lead to synaptic network dysfunction.
Assuntos
Moléculas de Adesão Celular/genética , Regulação da Expressão Gênica , Deficiência Intelectual/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Sinapses/metabolismo , Transmissão Sináptica/genética , Animais , Moléculas de Adesão Celular/metabolismo , Criança , Genótipo , Humanos , Deficiência Intelectual/metabolismo , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sinapses/genéticaRESUMO
Mutations of the Interleukin-1-receptor accessory protein like 1 (IL1RAPL1) gene are associated with cognitive impairment ranging from non-syndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of IL1/Toll receptors, which is localized at excitatory synapses and interacts with PSD-95. We previously showed that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun N-terminal kinase activity and PSD-95 phosphorylation. Here, we show that the IgG-like extracellular domains of IL1RAPL1 induce excitatory pre-synapse formation by interacting with protein tyrosine phosphatase delta (PTPδ). We also found that IL1RAPL1 TIR domains interact with RhoGAP2, which is localized at the excitatory post-synaptic density. More interestingly, the IL1RAPL1/PTPδ complex recruits RhoGAP2 at excitatory synapses to induce dendritic spine formation. We also found that the IL1RAPL1 paralog, IL1RAPL2, interacts with PTPδ and induces excitatory synapse and dendritic spine formation. The interaction of the IL1RAPL1 family of proteins with PTPδ and RhoGAP2 reveals a pathophysiological mechanism of cognitive impairment associated with a novel type of trans-synaptic signaling that regulates excitatory synapse and dendritic spine formation.
Assuntos
Quimerina 1/metabolismo , Genes Ligados ao Cromossomo X , Deficiência Intelectual/genética , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Sinapses/metabolismo , Animais , Células COS , Chlorocebus aethiops , Análise por Conglomerados , Espinhas Dendríticas/metabolismo , Células HEK293 , Humanos , Proteína Acessória do Receptor de Interleucina-1/química , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , RatosRESUMO
Oligophrenin-1 regulates dendritic spine morphology in the brain. Mutations in the oligophrenin-1 gene (OPHN1) cause intellectual disability. We discovered a previously unknown partner of oligophrenin-1, Rev-erbα, a nuclear receptor that represses the transcription of circadian oscillators. We found that oligophrenin-1 interacts with Rev-erbα in the mouse brain, causing it to locate to dendrites, reducing its repressor activity and protecting it from degradation. Our results indicate the presence of a circadian oscillator in the hippocampus, involving the clock gene Bmal1 (also known as Arntl), that is modulated by Rev-erbα and requires oligophrenin-1 for normal oscillation. We also found that synaptic activity induced Rev-erbα localization to dendrites and spines, a process that is mediated by AMPA receptor activation and requires oligophrenin-1. Our data reveal new interactions between synaptic activity and circadian oscillators, and delineate a new means of communication between nucleus and synapse that may provide insight into normal plasticity and the etiology of intellectual disability.
Assuntos
Relógios Circadianos/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Hipocampo/citologia , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Análise de Variância , Animais , Bicuculina/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Relógios Circadianos/genética , Inibidores de Cisteína Proteinase/farmacologia , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Dendritos/metabolismo , Interações Medicamentosas , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Humanos , Imunoprecipitação , Leupeptinas/farmacologia , Camundongos , Camundongos Knockout , Mutação/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Quinoxalinas/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Fatores de Tempo , Transfecção/métodos , Técnicas do Sistema de Duplo-Híbrido , Valina/análogos & derivados , Valina/farmacologiaRESUMO
BACKGROUND: Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) gene mutations are associated with cognitive impairment ranging from nonsyndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of Toll/IL-1 receptors, whose expression in the brain is upregulated by neuronal activity. Currently, very little is known about the function of this protein. We previously showed that IL1RAPL1 interacts with the neuronal calcium sensor NCS-1 and that it regulates voltage-gated calcium channel activity in PC12 cells. RESULTS: Here we show that IL1RAPL1 is present in dendritic spine where it interacts with PSD-95, a major component of excitatory postsynaptic compartment. Using gain- and loss-of-function experiments in neurons, we demonstrated that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun terminal kinase (JNK) activity and PSD-95 phosphorylation. Mice carrying a null mutation of the mouse Il1rapl1 gene show a reduction of both dendritic spine density and excitatory synapses in the CA1 region of the hippocampus. These structural abnormalities are associated with specific deficits in hippocampal long-term synaptic plasticity. CONCLUSION: The interaction of IL1RAPL1 with PSD-95 discloses a novel pathophysiological mechanism of cognitive impairment associated with alterations of the JNK pathway leading to a mislocalization of PSD-95 and abnormal synaptic organization and function.
Assuntos
Cognição , Proteína Acessória do Receptor de Interleucina-1/fisiologia , Mutação , Transdução de Sinais , Sinapses/metabolismo , Animais , Proteína 4 Homóloga a Disks-Large , Hipocampo/citologia , Hipocampo/metabolismo , Proteína Acessória do Receptor de Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Células PC12 , Fosforilação , RatosRESUMO
Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca(v)2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.
Assuntos
Colesterol/metabolismo , Exocitose/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Anticolesterolemiantes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Canais de Cálcio Tipo N/metabolismo , Células Cultivadas , Exocitose/efeitos dos fármacos , Fumonisinas/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Imunoglobulina G/metabolismo , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Microdomínios da Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Proteínas SNARE/metabolismo , Esfingolipídeos/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , Sinaptotagmina I/antagonistas & inibidores , Sinaptotagmina I/imunologia , Sinaptotagmina I/metabolismo , Ácidos Tricarboxílicos/farmacologiaRESUMO
The patho-physiological hypothesis of mental retardation caused by the deficiency of the RhoGAP Oligophrenin1 (OPHN1), relies on the well-known functions of Rho GTPases on neuronal morphology, i.e. dendritic spine structure. Here, we describe a new function of this Bin/Amphiphysin/Rvs domain containing protein in the control of clathrin-mediated endocytosis (CME). Through interactions with Src homology 3 domain containing proteins involved in CME, OPHN1 is concentrated to endocytic sites where it down-regulates the RhoA/ROCK signaling pathway and represses the inhibitory function of ROCK on endocytosis. Indeed disruption of Ophn1 in mice reduces the endocytosis of synaptic vesicles and the post-synaptic alpha-amino-3-hydroxy-5-methylisoazol-4-propionate (AMPA) receptor internalization, resulting in almost a complete loss of long-term depression in the hippocampus. Finally, pharmacological inhibition of this pathway by ROCK inhibitors fully rescued not only the CME deficit in OPHN1 null cells but also synaptic plasticity in the hippocampus from Ophn1 null model. Altogether, we uncovered a new patho-physiological mechanism for intellectual disabilities associated to mutations in RhoGTPases linked genes and also opened new directions for therapeutic approaches of congenital mental retardation.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Endocitose , Proteínas Ativadoras de GTPase/metabolismo , Deficiência Intelectual/fisiopatologia , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTPRESUMO
Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.