RESUMO
Autophagy is an intracellular catabolic process prominent in starvation, aging and disease. Neuronal autophagy is particularly important, as it affects the development and function of the nervous system, and is heavily implicated in neurodegenerative disease. Nonetheless, how autophagy is regulated in neurons remains poorly understood. Using an unbiased proteomics approach, we demonstrate that the primary initiator of autophagy, the UNC-51/ULK kinase, is negatively regulated by the ubiquitin ligase RPM-1. RPM-1 ubiquitin ligase activity restricts UNC-51 and autophagosome formation within specific axonal compartments, and exerts effects broadly across the nervous system. By restraining UNC-51 activity, RPM-1 inhibits autophagosome formation to affect axon termination, synapse maintenance and behavioral habituation. These results demonstrate how UNC-51 and autophagy are regulated subcellularly in axons, and unveils a mechanism for restricting initiation of autophagy across the nervous system. Our findings have important implications beyond nervous system development, given growing links between altered autophagy regulation and neurodegenerative diseases.
Assuntos
Autofagia/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Geneticamente Modificados , Autofagossomos/metabolismo , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Axônios/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Doenças Neurodegenerativas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Sinapses/genética , Sinapses/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Netrin and its receptor, Frazzled, dictate the strength of synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating gap junction localization in the presynaptic terminal. In Netrin mutant animals, the synaptic coupling between a giant interneuron and the "jump" motor neuron was weakened and dye coupling between these two neurons was severely compromised or absent. In cases in which Netrin mutants displayed apparently normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses and dye coupling between the giant fiber (GF) and the motor neuron was reduced or eliminated, suggesting that gap junctions were disrupted in the Netrin mutants. When we examined the gap junctions with antibodies to Shaking-B (ShakB) Innexin, they were significantly decreased or absent in the presynaptic terminal of the mutant GF. Frazzled loss of function mutants exhibited similar defects in synaptic transmission, dye coupling, and gap junction localization. These data are the first to show that Netrin and Frazzled regulate the placement of gap junctions presynaptically at a synapse.