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Molecular studies of synaptic vesicle recycling in health and disease

Final Report Summary - SYNAPSEFUNCTION (Molecular studies of synaptic vesicle recycling in health and disease)

Vesicle retrieval is critical to maintain the vesicle supply for neurotransmission and in this ERC project we have studied unresolved regulatory mechanisms that control the supply of novel vesicles for continued neurotransmission. Through genetic screens and phenotypic analyses we arrive at genes important for synaptic endocytosis and neurotransmission that are often implicated in human neurological disease. By analyzing the molecular mechanisms by which these genes control synaptic vesicle traffic we not only provide novel insights into synaptic function but also into the etiology of neurological disease.
During stimulation, vesicles are depleted and must be rapidly replenished by reformation at the synapse. Recent evidence implicates several Parkinson’s disease related proteins to affect pre-synaptic function, including LRRK2, but the exact molecular mechanisms at play remain enigmatic. We have studied the role of LRRK2, an evolutionary conserved kinase, at the synapse. Using genetic interactions and biochemical studies we found that LRRK2 affects synaptic function by phosphorylating EndoA, a key protein in endocytosis. We show that LRRK2-mediated EndoA phosphorylation is critical for normal EndoA function. Our findings may be interesting in light of Parkinson’s disease as EndoA is not only phosphorylated by LRRK2, but it also binds to Synaptojanin and is ubiquitinated by Parkin, two other proteins mutated in the disease as well. Hence, EndoA may reveal a new Parkinson-relevant pathway relevant for the regulation of presynaptic function.
Continued rounds of endo- and exocytosis may place stress on the vesicle cycle and old proteins would need to be removed. Given that synapses are often located far away from the cell body, we surmise that local mechanisms must exist. In an unbiased genetic screen for defects in synaptic transmission we identified a key and central pathway under control of Skywalker/TBC1D24 that restricts synaptic vesicles from traveling to endosomes. Skywalker activates the GTPase activity of Rab35 and expression of constitutive active Rab35 results in many vesicles to travel via an endosomal compartment. Using direct imaging of chimeric ubiquitinated synaptic vesicle proteins and flurescent timer-fusion proteins we show that when vesicles travel via an endosome (as is the case in skywalker mutants or in animals that express Rab35CA) this facilitates the replacement of dysfunctional synaptic vesicle components. This excessive endosomal trafficking then results in a more performant vesicle pool and a larger pool of vesicles that are ready for release. Our work suggests that trafficking of vesicles via endosomes and the degradation of dysfunctional components at lysosomes is a mechanism by which neurons regulate synaptic vesicle rejuvenation and neurotransmitter release.
Presynaptic terminals are essentially proteolipid machines and the synaptic vesicle membrane is continuously deformed. New synaptic vesicles are molted from the presynaptic membrane, and primed vesicles form a fusion pore and can collapse into the presynaptic membrane. In addition, turn over mechanisms such as autophagy or multivesicular body formation also rely on membrane deformation. To identify previously unstudied components involved in membrane deformation at synapses we conducted novel image based screens to identify the proteins involved in the regulation of membrane shape and synaptic vesicle recycling, identifying 204 new players. The identification of these new proteins, as well as our studies of the regulation of endocytosis impacts the information that is transmitted through neuronal circuits and potentially also neuronal health. Our ultimate hope is that these studies will not only provide valuable insights into the dynamics of the molecular mechanisms of presynaptic function, but they will further reveal mechanisms regulating synaptic plasticity and disease.