Een genetische screen identificeert de endo-lysosomale route als belangrijk mechanisme voor de vernieuwing van synaptische vesikel eiwitten
Genetic screens identifies endo-lysosomal pathway as a key regulator of synaptic vesicles rejuvenation
Fernandes, Ana Clara; S0218210
An efficient synaptic transmission is the basis for neuronal function. At the synapse vesicles filled with neurotransmitters are released in response to an action potential. These synapses are often located long distances away from the cell body and in order to maintain synaptic transmission synaptic vesicles are locally recycled. Understanding the mechanisms of vesicle recycling and synaptic transmission is key to understand how synaptic pathologies arise from synaptic dysfunction. In order to identify key players in synaptic transmission we have performed in the lab a forward genetic EMS screen. From a similar screen a novel key player in the synaptic vesicle cycle Skywalker - was identified. skywalker encodes an evolutionary conserved GTPase Activating Protein that inhibits Rab35 mediated synaptic vesicle trafficking in a pathway parallel to Rab5. Rab5 and Rab35 promote synaptic vesicles to fuse with endosomes and this additional trafficking step correlates with increased transmitter release and a larger pool of readily releasable vesicles (RRP). However, it is not clear how fusion of vesicles with endosomes results in increased neurotransmitter release.Recently it was reported that loss of function mutations in TBC1D24, the human homolog of skywalker, cause severe neurodegeneration, focal and infantile myoclonic epilepsy, malignant migrating partial seizures of infancy (MMPSI), intellectual disability and DOORS syndrome. However, the molecular nature of the defects upon loss of TBC1D24 remains poorly characterized. In this thesis I describe the different steps to validate next generation sequencing as a suitable approach for large-scale mapping of EMS induced mutants. One of the mutants identified with NGS is deep orange, an homolog of VPS18 that is a member of the HOPS complex, involved in endosome-to-lysosomal trafficking. The HOPS complex consists of Vps11, 16, 18, 33, 39 and 41 and binds to proteins that mediate fusion of cargo vesicles with the lysosome. Using a fluorescent synaptic vesicle-associated timer we show that the HOPS complex is critical for synaptic vesicle protein turnover, working downstream of Skywalker. Furthermore, similar to loss of TBC1D24 in humans, sky mutant flies show massive brain lesions reminiscent of neurodegeneration, which can also be rescued by the partial inhibition of Dor. We hypothesize that endosomes may serve as sorting stations for synaptic vesicle proteins, whereby dysfunctional synaptic vesicle proteins are removed from the vesicle cycle at endosomes and sent to the lysosome for degradation. When the dysfunctional vesicle proteins are removed from the vesicle cycle, synaptic vesicles would be populated by the remaining functional vesicle proteins, thus facilitating neurotransmission. Our work unveils a mechanism by which endosome-to-lysosomal trafficking controls synaptic vesicle protein turnover, ensuring proper levels of neurotransmitter release as well as neuronal survival in the context Sky/TBC1D24 function.