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1.
J Neurosci ; 41(35): 7329-7339, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34290081

RESUMO

Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1.SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.


Assuntos
Proteínas Munc18/metabolismo , Plasticidade Neuronal/fisiologia , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Animais , Feminino , Técnicas de Introdução de Genes , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Proteínas Munc18/deficiência , Mutação de Sentido Incorreto , Ésteres de Forbol/farmacologia , Fosforilação , Mutação Puntual , Proteína Quinase C/deficiência , Células de Purkinje/fisiologia , Proteínas Recombinantes/metabolismo , Transmissão Sináptica/efeitos dos fármacos
2.
J Neurochem ; 157(3): 450-466, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33259669

RESUMO

Loss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its target-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether loss of MUNC18-1 causes defects in intracellular membrane transport pathways in primary murine neurons that may explain neurodegeneration. Electron, confocal and super resolution microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall, ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. By synchronizing protein trafficking by conditional cargo retention in the endoplasmic reticulum using selective hooks (RUSH) and immunocytochemistry, we show that anterograde Endoplasmic Reticulum-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin B-subunit transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking in an antibody uptake assay. We conclude that MUNC18-1 deficient neurons have normal anterograde but reduced retrograde transport to the Golgi. The impairments in retrograde pathways suggest a role of MUNC18-1 in endosomal SNARE-dependent fusion and provide a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons.


Assuntos
Transporte Biológico/genética , Proteínas Munc18/deficiência , Proteínas Munc18/genética , Animais , Morte Celular , Membrana Celular/metabolismo , Células Cultivadas , Toxina da Cólera/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/patologia , Complexo de Golgi/ultraestrutura , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Knockout , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Neurônios/ultraestrutura , Proteínas SNARE/deficiência , Proteínas SNARE/genética
3.
Sci Rep ; 10(1): 3181, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081899

RESUMO

Phosphorylation of Munc18-1 (Stxbp1), a presynaptic organizer of synaptic vesicle fusion, is a powerful mechanism to regulate synaptic strength. Munc18-1 is a proposed substrate for the Down Syndrome-related kinase dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a) and mutations in both genes cause intellectual disability. However, the functional consequences of Dyrk1a-dependent phosphorylation of Munc18-1 for synapse function are unknown. Here, we show that the proposed Munc18-1 phosphorylation site, T479, is among the highly constrained phosphorylation sites in the coding regions of the gene and is also located within a larger constrained coding region. We confirm that Dyrk1a phosphorylates Munc18-1 at T479. Patch-clamp physiology in conditional null mutant hippocampal neurons expressing Cre and either wildtype, or mutants mimicking or preventing phosphorylation, revealed that synaptic transmission is similar among the three groups: frequency/amplitude of mEPSCs, evoked EPSCs, paired pulse plasticity, rundown kinetics upon intense activity and the readily releasable pool. However, synapses expressing the phosphomimic mutant responded to intense activity with more pronounced facilitation. These data indicate that Dyrk1a-dependent Munc18-1 phosphorylation has a minor impact on synaptic transmission, only after intense activity, and that the role of genetic variation in both genes in intellectual disability may be through different mechanisms.


Assuntos
Síndrome de Down/enzimologia , Proteínas Munc18/genética , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transmissão Sináptica , Animais , Sobrevivência Celular , Células HEK293 , Humanos , Camundongos , Proteínas Munc18/deficiência , Proteínas Munc18/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fases de Leitura Aberta/genética , Fosforilação , Treonina/metabolismo , Quinases Dyrk
4.
Nat Commun ; 10(1): 1917, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015467

RESUMO

STXBP1 and SCN2A gene mutations are observed in patients with epilepsies, although the circuit basis remains elusive. Here, we show that mice with haplodeficiency for these genes exhibit absence seizures with spike-and-wave discharges (SWDs) initiated by reduced cortical excitatory transmission into the striatum. Mice deficient for Stxbp1 or Scn2a in cortico-striatal but not cortico-thalamic neurons reproduce SWDs. In Stxbp1 haplodeficient mice, there is a reduction in excitatory transmission from the neocortex to striatal fast-spiking interneurons (FSIs). FSI activity transiently decreases at SWD onset, and pharmacological potentiation of AMPA receptors in the striatum but not in the thalamus suppresses SWDs. Furthermore, in wild-type mice, pharmacological inhibition of cortico-striatal FSI excitatory transmission triggers absence and convulsive seizures in a dose-dependent manner. These findings suggest that impaired cortico-striatal excitatory transmission is a plausible mechanism that triggers epilepsy in Stxbp1 and Scn2a haplodeficient mice.


Assuntos
Corpo Estriado/metabolismo , Proteínas Munc18/genética , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Neocórtex/metabolismo , Convulsões/genética , Transmissão Sináptica , Potenciais de Ação/efeitos dos fármacos , Animais , Anticonvulsivantes/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Dioxóis/farmacologia , Eletroencefalografia , Epilepsia Tipo Ausência/tratamento farmacológico , Epilepsia Tipo Ausência/genética , Epilepsia Tipo Ausência/metabolismo , Epilepsia Tipo Ausência/fisiopatologia , Etossuximida/farmacologia , Regulação da Expressão Gênica , Haploinsuficiência , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Interneurônios/patologia , Camundongos , Camundongos Knockout , Proteínas Munc18/deficiência , Canal de Sódio Disparado por Voltagem NAV1.2/deficiência , Neocórtex/efeitos dos fármacos , Neocórtex/patologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Piperidinas/farmacologia , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Convulsões/prevenção & controle , Transdução de Sinais , Tálamo/efeitos dos fármacos , Tálamo/metabolismo
5.
Cell Mol Gastroenterol Hepatol ; 6(4): 477-493.e1, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30364784

RESUMO

Background & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID. Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions. Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin-positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane. Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.


Assuntos
Diferenciação Celular , Enterócitos/patologia , Intestinos/patologia , Síndromes de Malabsorção/metabolismo , Microvilosidades/patologia , Mucolipidoses/metabolismo , Proteínas Munc18/deficiência , Proteínas Munc18/metabolismo , Organoides/metabolismo , Actinas/metabolismo , Animais , Núcleo Celular/metabolismo , Enterócitos/metabolismo , Humanos , Lisossomos/metabolismo , Síndromes de Malabsorção/patologia , Camundongos Knockout , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Mucolipidoses/patologia , Organoides/patologia , Organoides/ultraestrutura
6.
Eur Rev Med Pharmacol Sci ; 22(5): 1409-1414, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29565501

RESUMO

OBJECTIVE: Mucus production and hypersecretion are important pathophysiological features of asthma. Airway mucus secretion is more serious in obese asthma. Therefore, it is of great significance to elucidate the mechanism of asthma airway mucus high secretion in improving the control of asthma and the prognosis of obese asthmatic patients. MATERIALS AND METHODS: Obese asthmatic mice model was established to test the airway resistance and mucin secretion by hematoxylin-eosin (HE) staining. Munc18b and Muc5ac expression levels were determined by Western-blotting. Munc18b conditioned knockout mice were adopted to explore the mechanism of Muc5ac high secretion. RESULTS: The mice weight increased in obese asthmatic model accompanied by elevated airway resistance. HE staining showed enhanced mucin secretion, which was correlated to weight and airway resistance. Munc18b and Muc5ac expressions significant upregulated in an obese asthmatic mouse model compared with normal control. Muc5ac expression failed to show elevation in Munc18b conditioned knockout mice. CONCLUSIONS: Muc5ac high secretion was positively correlated with Munc18b upregulation in obese asthma. Munc18b participated in inducing Muc5ac high expression.


Assuntos
Asma/patologia , Mucina-5AC/metabolismo , Proteínas Munc18/metabolismo , Obesidade/patologia , Animais , Asma/etiologia , Asma/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Munc18/deficiência , Proteínas Munc18/genética , Obesidade/complicações , Obesidade/metabolismo , Ovalbumina/imunologia , Regulação para Cima
7.
Cereb Cortex ; 28(7): 2253-2266, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28520937

RESUMO

Endocannabinoids (ECBs) depress transmitter release at sites throughout the brain. Here, we describe another form of ECB signaling that triggers a novel form of long-term potentiation (LTP) localized to the lateral perforant path (LPP) which conveys semantic information from cortex to hippocampus. Two cannabinoid CB1 receptor (CB1R) signaling cascades were identified in hippocampus. The first is pregnenolone sensitive, targets vesicular protein Munc18-1 and depresses transmitter release; this cascade is engaged by CB1Rs in Schaffer-Commissural afferents to CA1 but not in the LPP, and it does not contribute to LTP. The second cascade is pregnenolone insensitive and LPP specific; it entails co-operative CB1R/ß1-integrin signaling to effect synaptic potentiation via stable enhancement of transmitter release. The latter cascade is engaged during LPP-dependent learning. These results link atypical ECB signaling to the encoding of a fundamental component of episodic memory and suggest a novel route whereby endogenous and exogenous cannabinoids affect cognition.


Assuntos
Córtex Cerebral/fisiologia , Endocanabinoides/metabolismo , Hipocampo/fisiologia , Memória/fisiologia , Vias Neurais/fisiologia , Transdução de Sinais/fisiologia , Animais , Inibidores Enzimáticos/farmacologia , GABAérgicos/farmacologia , Hipocampo/citologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Munc18/deficiência , Proteínas Munc18/genética , Vias Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Transtornos da Percepção/genética , Transtornos da Percepção/patologia , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
8.
J Proteome Res ; 16(10): 3787-3804, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28792770

RESUMO

Clinical trials have been conducted for the neuronal ceroid lipofuscinoses (NCLs), a group of neurodegenerative lysosomal diseases that primarily affect children. Whereas clinical rating systems will evaluate long-term efficacy, biomarkers to measure short-term response to treatment would be extremely valuable. To identify candidate biomarkers, we analyzed autopsy brain and matching CSF samples from controls and three genetically distinct NCLs due to deficiencies in palmitoyl protein thioesterase 1 (CLN1 disease), tripeptidyl peptidase 1 (CLN2 disease), and CLN3 protein (CLN3 disease). Proteomic and biochemical methods were used to analyze lysosomal proteins, and, in general, we find that changes in protein expression compared with control were most similar between CLN2 disease and CLN3 disease. This is consistent with previous observations of biochemical similarities between these diseases. We also conducted unbiased proteomic analyses of CSF and brain using isobaric labeling/quantitative mass spectrometry. Significant alterations in protein expression were identified in each NCL, including reduced STXBP1 in CLN1 disease brain. Given the confounding variable of post-mortem changes, additional validation is required, but this study provides a useful starting set of candidate NCL biomarkers for further evaluation.


Assuntos
Encéfalo/metabolismo , Proteínas Munc18/genética , Lipofuscinoses Ceroides Neuronais/genética , Proteômica , Aminopeptidases/deficiência , Aminopeptidases/genética , Autopsia , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/química , Biomarcadores/metabolismo , Encéfalo/patologia , Líquido Cefalorraquidiano/química , Líquido Cefalorraquidiano/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/deficiência , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Humanos , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Proteínas Munc18/deficiência , Mutação , Lipofuscinoses Ceroides Neuronais/líquido cefalorraquidiano , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Serina Proteases/deficiência , Serina Proteases/genética , Tioléster Hidrolases/deficiência , Tioléster Hidrolases/genética , Tripeptidil-Peptidase 1
9.
J Neurosci ; 36(2): 561-76, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26758845

RESUMO

The role of synaptic activity during early formation of neural circuits is a topic of some debate; genetic ablation of neurotransmitter release by deletion of the Munc18-1 gene provides an excellent model to answer the question of whether such activity is required for early circuit formation. Previous analysis of Munc18-1(-/-) mouse mutants documented their grossly normal nervous system, but its molecular differentiation has not been assessed. Munc18-1 deletion in mice also results in widespread neurodegeneration that remains poorly characterized. In this study, we demonstrate that the early stages of spinal motor circuit formation, including motor neuron specification, axon growth and pathfinding, and mRNA expression, are unaffected in Munc18-1(-/-) mice, demonstrating that synaptic activity is dispensable for early nervous system development. Furthermore, we show that the neurodegeneration caused by Munc18-1 loss is cell autonomous, consistent with apparently normal expression of several neurotrophic factors and normal GDNF signaling. Consistent with cell-autonomous degeneration, we demonstrate defects in the trafficking of the synaptic proteins Syntaxin1a and PSD-95 and the TrkB and DCC receptors in Munc18-1(-/-) neurons; these defects do not appear to cause ER stress, suggesting other mechanisms for degeneration. Finally, we demonstrate pathological similarities to Alzheimer's disease, such as altered Tau phosphorylation, neurofibrillary tangles, and accumulation of insoluble protein plaques. Together, our results shed new light upon the neurodegeneration observed in Munc18-1(-/-) mice and argue that this phenomenon shares parallels with neurodegenerative diseases. SIGNIFICANCE STATEMENT: In this work, we demonstrate the absence of a requirement for regulated neurotransmitter release in the assembly of early neuronal circuits by assaying transcriptional identity, axon growth and guidance, and mRNA expression in Munc18-1-null mice. Furthermore, we characterize the neurodegeneration observed in Munc18-1 mutants and demonstrate that this cell-autonomous process does not appear to be a result of defects in growth factor signaling or ER stress caused by protein trafficking defects. However, we find the presence of various pathological hallmarks of Alzheimer's disease that suggest parallels between the degeneration in these mutants and neurodegenerative conditions.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios Motores/patologia , Proteínas Munc18/deficiência , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Medula Espinal/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Morte Celular/genética , Receptor DCC , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Guanilato Quinases/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Munc18/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Transporte Proteico/genética , Receptor trkB/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/genética , Medula Espinal/embriologia , Sintaxina 1/metabolismo , Proteínas Supressoras de Tumor/metabolismo
10.
J Neurochem ; 122(5): 1081-91, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22765017

RESUMO

Dual-specificity tyrosine(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) is a protein kinase that might be responsible for mental retardation and early onset of Alzheimer's disease in Down's syndrome patients. Dyrk1A plays a role in many cellular pathways through phosphorylation of diverse substrate proteins; however, its role in synaptic vesicle exocytosis is poorly understood. Munc18-1, a central regulator of neurotransmitter release, interacts with Syntaxin 1 and X11α. Syntaxin 1 is a key soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein involved in synaptic vesicle docking/fusion events, and X11α modulates amyloid precursor protein processing and ß amyloid generation. In this study, we demonstrate that Dyrk1A interacts with and phosphorylates Munc18-1 at the Thr(479) residue. The phosphorylation of Munc18-1 at Thr(479) by Dyrk1A stimulated binding of Munc18-1 to Syntaxin 1 and X11α. Furthermore, the levels of phospho-Thr(479) -Munc18-1 were enhanced in the brains of transgenic mice over-expressing Dyrk1A protein, providing in vivo evidence of Munc18-1 phosphorylation by Dyrk1A. These results reveal a link between Munc18-1 and Dyrk1A in synaptic vesicle trafficking and amyloid precursor protein processing, suggesting that up-regulated Dyrk1A in Down's syndrome and Alzheimer's disease brains may contribute to some pathological features, including synaptic dysfunction and cognitive defect through abnormal phosphorylation of Munc18-1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sintaxina 1/metabolismo , Trifosfato de Adenosina/farmacocinética , Animais , Encéfalo/metabolismo , Linhagem Celular Transformada , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Proteínas Munc18/deficiência , Proteínas Munc18/genética , Mutação/fisiologia , Isótopos de Fósforo/farmacocinética , Fosforilação/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/farmacologia , Treonina/metabolismo , Transfecção , Quinases Dyrk
11.
Biol Cell ; 102(8): 479-88, 2010 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-20497124

RESUMO

BACKGROUND INFORMATION: During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth. RESULTS: We tested this possibility in dissociated cell cultures and organotypic slice cultures of two release-deficient mice (Munc18-1 null and Munc13-1/2 double null). Both types of release-deficient neurons have a decreased outgrowth speed and therefore have a smaller total neurite length during early development [DIV1-4 (day in vitro 1-4)]. In addition, more filopodia per growth cone were observed in Munc18-1 null, but not WT (wild-type) or Munc13-1/2 double null neurons. The smaller total neurite length during early development was no longer observed after synaptogenesis (DIV14-23). CONCLUSION: These data suggest that the inability of vesicle fusion in the growth cone affects outgrowth during the initial phases when outgrowth speed is high, but not during/after synaptogenesis. Overall, the outgrowth speed is probably not rate-limiting during neuronal network formation, at least in vitro. In addition, Munc18, but not Munc13, regulates growth cone filopodia, potentially via its previously observed effect on filamentous actin.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Munc18/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuritos/fisiologia , Animais , Cones de Crescimento/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos , Camundongos Transgênicos , Proteínas Munc18/deficiência , Proteínas do Tecido Nervoso/deficiência , Pseudópodes/metabolismo
12.
J Clin Invest ; 119(12): 3765-73, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19884660

RESUMO

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous autosomal recessive immune disorder characterized by the occurrence of uncontrolled activation of lymphocytes and macrophages infiltrating multiple organs. Disease-causing mutations in the perforin (PRF1; also known as FHL2), Munc13-4 (UNC13D; also known as FHL3), and syntaxin-11 (STX11; also known as FHL4) genes have been identified in individuals with FHL. These genes all encode proteins involved in the cytotoxic activity of lymphocytes. Here, we show that the gene encoding syntaxin-binding protein 2 (Munc18-2; official gene symbol STXBP2) is mutated in another subset of patients with FHL (designated by us as "FHL5"). Lymphoblasts isolated from these patients had strongly decreased STXBP2 protein expression, and NK cells exhibited impaired cytotoxic granule exocytosis, a defect that could be overcome by ectopic expression of wild-type STXBP2. Furthermore, we provide evidence that syntaxin-11 is the main partner of STXBP2 in lymphocytes, as its expression required the presence of STXBP2. Our work shows that STXBP2 deficiency causes FHL5. These data indicate that STXBP2 is required at a late step of the secretory pathway for the release of cytotoxic granules by binding syntaxin 11, another component of the intracellular membrane fusion machinery.


Assuntos
Células Matadoras Naturais/fisiologia , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/fisiopatologia , Proteínas Munc18/deficiência , Proteínas Munc18/genética , Adolescente , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Criança , Consanguinidade , Exocitose/genética , Exocitose/fisiologia , Feminino , Genes Recessivos , Homozigoto , Humanos , Lactente , Íntrons , Linfo-Histiocitose Hemofagocítica/classificação , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Munc18/química , Proteínas Munc18/fisiologia , Mutação de Sentido Incorreto , Linhagem , Proteínas Qa-SNARE/fisiologia , Sítios de Splice de RNA , Homologia de Sequência de Aminoácidos , Adulto Jovem
13.
Diabetes ; 58(5): 1165-74, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19188424

RESUMO

OBJECTIVE: The Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4-mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mechanism(s) using the islet beta-cell as a model system. RESEARCH DESIGN AND METHODS: Perifusion analyses of isolated Munc18c- (-/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 beta-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/fusion of vesicle-associated membrane protein (VAMP)2-containing insulin granules. Electron microscopy was used to gauge changes in granule localization. RESULTS: Munc18c (-/+) islets secreted approximately 60% less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (-/+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2-Syntaxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion. CONCLUSIONS: Collectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion.


Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Ilhotas Pancreáticas/metabolismo , Proteínas Munc18/genética , Proteína 2 Associada à Membrana da Vesícula/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Primers do DNA , Diabetes Mellitus Tipo 2/genética , Exocitose , Feminino , Inativação Gênica , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Munc18/deficiência , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Transfecção
14.
Mol Immunol ; 44(13): 3427-33, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17408745

RESUMO

Recent studies have revealed that SNARE proteins are involved in exocytotic granular content release in mast cells as well as in neurotransmitter release in neural cells. However, the proteins that regulate the structure and activity of SNARE proteins in mast cells are not well understood. Munc18 is one such regulatory protein that plays a crucial role in neurotransmitter release. In this study, we investigated the role of Munc18 and its mechanism for regulating exocytotic release (degranulation) in rat basophilic leukemia cells (RBL-2H3). We obtained RBL-2H3 cells that express a low level of Munc18-2 and found that degranulation was remarkably inhibited in knockdown cells without any change in the expression level of syntaxins or Ca(2+) mobilization. We also observed the behavior of secretory granules in a single cell, and found no significant changes in their number and distribution in Munc18-2 knockdown cells. Using chimera proteins fused with fluorescent proteins, we demonstrated that Munc18-2 interacted with syntaxin-3, but not with syntaxin-4, in vivo. Interestingly, this interaction occurred not only on plasma membrane but also on secretory granules, suggesting that Munc18-2 may regulate granule-granule fusion as well as granule-plasma membrane fusion. These observations suggest that Munc18-2 together with syntaxin-3 regulate degranulation positively during the process of membrane fusion between secretory granules and plasma membrane, rather than during processes that regulate the number or behavior of secretory granules.


Assuntos
Degranulação Celular/fisiologia , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Proteínas Munc18/fisiologia , Proteínas Qa-SNARE/metabolismo , Animais , Degranulação Celular/genética , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Exocitose/genética , Fusão de Membrana/genética , Proteínas Munc18/deficiência , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/fisiologia , Ratos , Vesículas Secretórias/genética , Vesículas Secretórias/metabolismo , Vesículas Secretórias/fisiologia
15.
Nat Neurosci ; 10(3): 340-7, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17259982

RESUMO

Spontaneous activity generated in the retina is necessary to establish a precise retinotopic map, but the underlying mechanisms are poorly understood. We demonstrate here that neural activity controls ephrin-A-mediated responses. In the mouse retinotectal system, we show that spontaneous activity of the retinal ganglion cells (RGCs) is needed, independently of synaptic transmission, for the ordering of the retinotopic map and the elimination of exuberant retinal axons. Activity blockade suppressed the repellent action of ephrin-A on RGC growth cones by cyclic AMP (cAMP)-dependent pathways. Unexpectedly, the ephrin-A5-induced retraction required cAMP oscillations rather than sustained increases in intracellular cAMP concentrations. Periodic photo-induced release of caged cAMP in growth cones rescued the response to ephrin-A5 when activity was blocked. These results provide a direct molecular link between spontaneous neural activity and axon guidance mechanisms during the refinement of neural maps.


Assuntos
AMP Cíclico/metabolismo , Periodicidade , Retina/fisiologia , Transdução de Sinais/fisiologia , Vias Visuais/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Embrião de Mamíferos , Efrina-A5/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/fisiologia , Camundongos , Camundongos Knockout , Proteínas Munc18/deficiência , Técnicas de Cultura de Órgãos , Inibidores de Fosfodiesterase/farmacologia , Quinoxalinas/farmacologia , Retina/citologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/fisiologia , Tetrodotoxina/farmacologia , Vias Visuais/citologia
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