RESUMO
OBJECTIVE: Mutations in ABCD1 cause the neurodegenerative disease, adrenoleukodystrophy, which manifests as the spinal cord axonopathy adrenomyeloneuropathy (AMN) in nearly all males surviving into adulthood. Microglial dysfunction has long been implicated in pathogenesis of brain disease, but its role in the spinal cord is unclear. METHODS: We assessed spinal cord microglia in humans and mice with AMN and investigated the role of ABCD1 in microglial activity toward neuronal phagocytosis in cell culture. Because mutations in ABCD1 lead to incorporation of very-long-chain fatty acids into phospholipids, we separately examined the effects of lysophosphatidylcholine (LPC) upon microglia. RESULTS: Within the spinal cord of humans and mice with AMN, upregulation of several phagocytosis-related markers, such as MFGE8 and TREM2, precedes complement activation and synapse loss. Unexpectedly, this occurs in the absence of overt inflammation. LPC C26:0 added to ABCD1-deficient microglia in culture further enhances MFGE8 expression, aggravates phagocytosis, and leads to neuronal injury. Furthermore, exposure to a MFGE8-blocking antibody reduces phagocytic activity. INTERPRETATION: Spinal cord microglia lacking ABCD1 are primed for phagocytosis, affecting neurons within an altered metabolic milieu. Blocking phagocytosis or specific phagocytic receptors may alleviate synapse loss and axonal degeneration. Ann Neurol 2017;82:813-827.
Assuntos
Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/fisiologia , Adrenoleucodistrofia/fisiopatologia , Microglia/fisiologia , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Anticorpos/imunologia , Antígenos de Superfície/biossíntese , Antígenos de Superfície/imunologia , Estudos de Casos e Controles , Células Cultivadas , Técnicas de Cocultura , Expressão Gênica/efeitos dos fármacos , Humanos , Lisofosfatidilcolinas/farmacologia , Glicoproteínas de Membrana/biossíntese , Camundongos Knockout , Microglia/efeitos dos fármacos , Proteínas do Leite/biossíntese , Proteínas do Leite/imunologia , Neurônios/fisiologia , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Cultura Primária de Células , Receptores Imunológicos/biossíntese , Medula Espinal/fisiologiaRESUMO
Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans , Músculos/fisiologiaRESUMO
Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1, and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Mutação/genética , Vesículas Secretórias/metabolismo , Proteína rab2 de Ligação ao GTP/metabolismo , Análise de Variância , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Mapeamento Cromossômico , Clonagem Molecular , Endossomos/metabolismo , Endossomos/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Transporte Proteico , Vesículas Secretórias/genética , Vesículas Secretórias/ultraestrutura , Proteína rab2 de Ligação ao GTP/genéticaRESUMO
Neurons secrete neuropeptides from dense core vesicles (DCVs) to modulate neuronal activity. Little is known about how neurons manage to differentially regulate the release of synaptic vesicles (SVs) and DCVs. To analyze this, we screened all Caenorhabditis elegans Rab GTPases and Tre2/Bub2/Cdc16 (TBC) domain containing GTPase-activating proteins (GAPs) for defects in DCV release from C. elegans motoneurons. rab-5 and rab-10 mutants show severe defects in DCV secretion, whereas SV exocytosis is unaffected. We identified TBC-2 and TBC-4 as putative GAPs for RAB-5 and RAB-10, respectively. Multiple Rabs and RabGAPs are typically organized in cascades that confer directionality to membrane-trafficking processes. We show here that the formation of release-competent DCVs requires a reciprocal exclusion cascade coupling RAB-5 and RAB-10, in which each of the two Rabs recruits the other's GAP molecule. This contributes to a separation of RAB-5 and RAB-10 domains at the Golgi-endosomal interface, which is lost when either of the two GAPs is inactivated. Taken together, our data suggest that RAB-5 and RAB-10 cooperate to locally exclude each other at an essential stage during DCV sorting.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Neurônios Motores/metabolismo , Neuropeptídeos/metabolismo , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Transporte Biológico/fisiologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Endossomos/genética , Endossomos/metabolismo , Exocitose/fisiologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Mutação , Vesículas Secretórias/genética , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.