RESUMO
The G protein-coupled receptor 17 (GPR17) plays crucial roles in myelination. It is highly expressed during transition of oligodendrocyte progenitor cells to immature oligodendrocytes, but, after this stage, it must be down-regulated to allow generation of mature myelinating cells. After endocytosis, GPR17 is sorted into lysosomes for degradation or recycled to the plasma membrane. Balance between degradation and recycling is important for modulation of receptor levels at the cell surface and thus for the silencing/activation of GPR17-signaling pathways that, in turn, affect oligodendrocyte differentiation. The molecular mechanisms at the basis of these processes are still partially unknown and their characterization will allow a better understanding of myelination and provide cues to interpret the consequences of GPR17 dysfunction in diseases. Here, we demonstrate that the endocytic trafficking of GPR17 is mediated by the interaction of a type I PDZ-binding motif located at the C-terminus of the receptor and SNX27, a recently identified protein of the endosome-associated retromer complex and whose functions in oligodendrocytes have never been studied. SNX27 knock-down significantly reduces GPR17 plasma membrane recycling in differentiating oligodendrocytes while accelerating cells' terminal maturation. Interestingly, trisomy-linked down-regulation of SNX27 expression in the brain of Ts65Dn mice, a model of Down syndrome, correlates with a decrease in GPR17(+) cells and an increase in mature oligodendrocytes, which, however, fail in reaching full maturation, eventually leading to hypomyelination. Our data demonstrate that SNX27 modulates GPR17 plasma membrane recycling and stability, and that disruption of the SNX27/GPR17 interaction might contribute to pathological oligodendrocyte differentiation defects. GLIA 2016. GLIA 2016;64:1437-1460.
Assuntos
Transporte Biológico/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Nexinas de Classificação/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/patologia , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Endocitose/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligodendroglia/patologia , Nexinas de Classificação/deficiência , Nexinas de Classificação/genéticaRESUMO
Mutations in the CACNA1A gene, which encodes the pore-forming α1A subunit of the CaV2.1 voltage-gated calcium channel, cause a number of human neurologic diseases including familial hemiplegic migraine. We have analyzed the functional impact of the E1015K amino acid substitution located in the "synprint" domain of the α1A subunit. This variant was identified in two families with hemiplegic migraine and in one patient with migraine with aura. The wild type (WT) and the E1015K forms of the GFP-tagged α1A subunit were expressed in cultured hippocampal neurons and HEK cells to understand the role of the variant in the transport activity and physiology of CaV2.1. The E1015K variant does not alter CaV2.1 protein expression, and its transport to the cell surface and synaptic terminals is similar to that observed for WT channels. Electrophysiological data demonstrated that E1015K channels have increased current density and significantly altered inactivation properties compared with WT. Furthermore, the SNARE proteins syntaxin 1A and SNAP-25 were unable to modulate voltage-dependent inactivation of E1015K channels. Overall, our findings describe a genetic variant in the synprint site of the CaV2.1 channel which is characterized by a gain-of-function and associated with both hemiplegic migraine and migraine with aura in patients.
Assuntos
Canais de Cálcio Tipo N , Hipocampo , Enxaqueca com Aura , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso , Terminações Pré-Sinápticas , Adolescente , Adulto , Substituição de Aminoácidos , Animais , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Criança , Feminino , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Transporte de Íons/genética , Masculino , Pessoa de Meia-Idade , Enxaqueca com Aura/genética , Enxaqueca com Aura/metabolismo , Enxaqueca com Aura/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Coelhos , Ratos , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMO
GPR17 is a G-protein-coupled receptor that is activated by two classes of molecules: uracil-nucleotides and cysteinyl-leukotrienes. GPR17 is required for initiating the differentiation of oligodendrocyte precursors but has to be down-regulated to allow cells to undergo terminal maturation. Although a great deal has been learned about GPR17 expression and signaling, no information is currently available about the trafficking of native receptors after the exposure of differentiating oligodendrocytes to endogenous agonists. Here, we demonstrate that neuron-conditioned medium induces the transcriptionally mediated, time-regulated expression of GPR17 in Oli-neu, an oligodendrocyte precursor cell line, making these cells suitable for studying the endocytic traffic of the native receptor. Agonist-induced internalization, intracellular trafficking, and membrane recycling of GPR17 were analyzed by biochemical and immunofluorescence assays using an ad hoc-developed antibody against the extracellular N-terminal of GPR17. Both UDP-glucose and LTD(4) increased GPR17 internalization, although with different efficiency. At early time points, internalized GPR17 co-localized with transferrin receptor, whereas at later times it partially co-localized with the lysosomal marker Lamp1, suggesting that a portion of GPR17 is targeted to lysosomes upon ligand binding. An analysis of receptor recycling and degradation demonstrated that a significant aliquot of GPR17 is recycled to the cell surface. Furthermore, internalized GPR17 displayed a co-localization with the marker of the "short loop" recycling endosomes, Rab4, while showing very minor co-localization with the "long loop" recycling marker, Rab11. Our results provide the first data on the agonist-induced trafficking of native GPR17 in oligodendroglial cells and may have implications for both physiological and pathological myelination.
Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Clonagem Molecular , Endocitose , Humanos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Oligodendroglia/citologia , Interferência de RNA , 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
LIPIDS MAY AFFECT SYNAPTIC FUNCTION IN AT LEAST TWO WAYS: by acting as ligands for effector proteins [e.g., phosphatidylinositol (4,5) bisphosphate, diacylglycerol-mediated signaling] or by modifying the physicochemical properties and molecular organization of synaptic membranes. One that acts in the latter manner is cholesterol, an essential structural component of plasma membranes that is largely enriched in the membranes of synapses and synaptic vesicles, in which it may be involved in lipid-lipid and protein-lipid interactions. Cholesterol is an important constituent of the "membrane rafts" that may play a role in recruiting and organizing the specific proteins of the exocytic pathways. Furthermore, many synaptic proteins bind directly to cholesterol. The regulation of cholesterol and lipid levels may therefore influence the specific interactions and activity of synaptic proteins, and have a strong impact on synaptic functions.