RESUMO
Genetic mutations in TBC1D24 have been associated with multiple phenotypes, with epilepsy being the main clinical manifestation. The TBC1D24 protein consists of the unique association of a Tre2/Bub2/Cdc16 (TBC) domain and a TBC/lysin motif domain/catalytic (TLDc) domain. More than 50 missense and loss-of-function mutations have been described and are spread over the entire protein. Through whole genome/exome sequencing we identified compound heterozygous mutations, R360H and G501R, within the TLDc domain, in an index family with a Rolandic epilepsy exercise-induced dystonia phenotype (http://omim.org/entry/608105). A 20-year long clinical follow-up revealed that epilepsy was self-limited in all three affected patients, but exercise-induced dystonia persisted into adulthood in two. Furthermore, we identified three additional sporadic paediatric patients with a remarkably similar phenotype, two of whom had compound heterozygous mutations consisting of an in-frame deletion I81_K84 and an A500V mutation, and the third carried T182M and G511R missense mutations, overall revealing that all six patients harbour a missense mutation in the subdomain of TLDc between residues 500 and 511. We solved the crystal structure of the conserved Drosophila TLDc domain. This allowed us to predict destabilizing effects of the G501R and G511R mutations and, to a lesser degree, of R360H and potentially A500V. Next, we characterized the functional consequences of a strong and a weak TLDc mutation (TBC1D24G501R and TBC1D24R360H) using Drosophila, where TBC1D24/Skywalker regulates synaptic vesicle trafficking. In a Drosophila model neuronally expressing human TBC1D24, we demonstrated that the TBC1D24G501R TLDc mutation causes activity-induced locomotion and synaptic vesicle trafficking defects, while TBC1D24R360H is benign. The neuronal phenotypes of the TBC1D24G501R mutation are consistent with exacerbated oxidative stress sensitivity, which is rescued by treating TBC1D24G501R mutant animals with antioxidants N-acetylcysteine amide or α-tocopherol as indicated by restored synaptic vesicle trafficking levels and sustained behavioural activity. Our data thus show that mutations in the TLDc domain of TBC1D24 cause Rolandic-type focal motor epilepsy and exercise-induced dystonia. The humanized TBC1D24G501R fly model exhibits sustained activity and vesicle transport defects. We propose that the TBC1D24/Sky TLDc domain is a reactive oxygen species sensor mediating synaptic vesicle trafficking rates that, when dysfunctional, causes a movement disorder in patients and flies. The TLDc and TBC domain mutations' response to antioxidant treatment we observed in the animal model suggests a potential for combining antioxidant-based therapeutic approaches to TBC1D24-associated disorders with previously described lipid-altering strategies for TBC domain mutations.
Assuntos
Acetilcisteína/análogos & derivados , Antioxidantes/uso terapêutico , Modelos Animais de Doenças , Drosophila melanogaster/fisiologia , Distonia/tratamento farmacológico , Epilepsia Rolândica/genética , Proteínas Ativadoras de GTPase/genética , Esforço Físico , alfa-Tocoferol/uso terapêutico , Acetilcisteína/uso terapêutico , Adolescente , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Transporte Biológico/efeitos dos fármacos , Domínio Catalítico/genética , Criança , Pré-Escolar , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Distonia/etiologia , Epilepsia Rolândica/tratamento farmacológico , Feminino , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/fisiologia , Humanos , Lactente , Locomoção/genética , Locomoção/fisiologia , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Neurônios/fisiologia , Estresse Oxidativo , Linhagem , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Vesículas Sinápticas/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P2, but this function appears normal in SynaptojaninRQ knock-in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P2, two lipids found in autophagosomal membranes. Using advanced imaging, we show that SynaptojaninRQ mutants accumulate the PI(3)P/PI(3,5)P2-binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell-derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in SynaptojaninRQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic-specific autophagy defects to Parkinson's disease.