Molecular basis for the role of EndoA1 and SGIP1 in synaptic homeostasis and Parkinson's disease

Korte inhoud:Synapses are specialized neuronal compartment responsible for the communication between neurons. Their health is challenged by the accumulation of dysfunctional proteins and organelles as a result of the high firing frequency neurons sustain throughout the lifetime of an organism. Homeostatic processes are in place to maintain synaptic health and function. Among these is autophagy. Several studies have shown that autophagy at synapses is regulated by synapse-resident proteins, suggesting that this pathway at these locations is differentially regulated than autophagy in other compartments. Autophagy induction at synapses has been shown to be linked to neurotransmission, but how this cross-regulation is achieved is just beginning to be unraveled. By performing live imaging in Drosophila NMJs, we show that extracellular Ca2+ influx into the pre-synaptic compartment is necessary and sufficient to induce autophagosome formation. Moreover, we demonstrated that this effect is mediated by the endocytic protein EndoA and can be modulated by mutations in the disordered loop region of EndoA that mimic the presence or absence of Ca2+. By performing a biophysical examination of recombinant EndoA mutants, we found that the mutations affect EndoA intrinsic structural dynamics and that they do so in an opposite way: the mutant resembling the absence of Ca2+ is more rigid, while the mutant mimicking the presence of Ca2+ in more flexible. In vivo super-resolution imaging revealed that EndoA gains mobility when the Ca2+ concentration is high. This ability allows the protein to diffuse from nanodomain at the plasma membrane -where it resides in basal condition- to the lumen of the synapse, where it drives autophagosome formation. EndoA participates also in starvation-induced autophagy. Interestingly, we observed that starvation and Ca2+ influx are distinct triggers that induce two parallel pathways of autophagy induction, both resulting in the diffusion of EndoA from the periphery to the lumen of the synapse. Several studies have shown that dysregulation of synaptic autophagy, either too much or too little, is deleterious for neurons' survival. We also show in our models that this is the case. This is relevant as several proteins mutated in PD have a role in synaptic autophagy. We characterized, for the first time, a candidate PD variant on EndoA disordered region and found that this mutation blocks autophagy in Drosophila NMJs and in neurites of differentiated dopaminergic neurons. The mutation does so by abolishing the diffusion of EndoA to the lumen of the synapse. In fact, the protein shows low mobility and stays preferentially confined in nanodomains at the plasma membrane. Our findings provide evidence for a role of synaptic homeostasis in neuronal demise and PD. Additionally, we show that autophagy induction is tightly linked to neuronal activity. The latter was shown to also be affected by mutations in genes associated with PD pathophysiology. We report for the first time an association between the EndoA1 interactor SGIP1 and parkinsonism. We modelled the candidate pathogenic mutation in Drosophila and iPSC-derived dopaminergic neurons and found that the mutation destabilizes the protein. Electrophysiological and ultrastructural characterization of dSgip1 loss-of-function Drosophila synapses revelated that evoked neurotransmission is impaired in this model, which also display a significant reduction in the size of SVs and lack MVBs. These phenotypes are accompanied by a reduction in Syt1 protein levels. Moreover, dSgip1 loss-of-function flies show severe behavioral deficits and generalized neurodegeneration, supporting SGIP1 as a novel candidate PD gene. Hence, unbalanced neurotransmission and synaptic homeostasis contribute to the neuronal degeneration observed in PD.

Jaar van publicatie:2023

Toegankelijkheid:Open