Membrane Traffic Defects in Parkinson Disease – MeTDePaDi

Parkinson’s disease (PD) is a major progressive neurodegenerative disorder affecting several regions of the brain, especially the substantia nigra (SN). Although symptomatic treatments are available, there are no protective or curative treatments and diagnosis is established only when degeneration is well advanced. There is thus a major medical need to develop disease modifying treatment and biomarkers of early stage PD. In this regard, genetics has revealed a-synuclein, a protein enriched in Lewy bodies of PD brains, and LRRK2, a complex signalling protein, as major disease factors involved in both familial and sporadic PD. Recent data suggest that a-synuclein, localized in presynaptic nerve terminals, regulates the function of SNAREs, the core machinery mediating intracellular membrane fusion and traffic. VAMP7 is a v-SNARE related to synaptic VAMP2 and is enriched in neuronal cells particularly in 1) somatodendritic compartment of SN neurons where dopaminergic (DA) neurons affected in PD are located and 2) nerve terminals where neuronal activity is regulated by DA released by dendrites of SN neurons and 3) the striatum where DA neurons project. Growing evidence also shows a role for LRRK2 and its close homolog LRRK1 in autophagy, and inhibition of LRRK2 induces protective autophagy. Recently VAMP7 was shown to interact with LRRK1 and a-synuclein to bind to VAMP2 and block synaptic SNARE-dependent vesicle fusion.
These observations led us to the working hypothesis that VAMP7-dependent organelle biogenesis and exocytosis in brain may be regulated by LRRK2 and a-synuclein and that this mechanism may be defective in PD. The important physiopathological implication would be that early PD symptoms may result from defective vesicular homeostasis mediated by VAMP7. Modulating VAMP7-dependent secretion may thus be a new promising target for treating early-diagnosed PD patients.
Our main objective is therefore to characterize the structure/function of the biochemical connections between PD-related genes LRRK2 and a-synuclein in VAMP7-dependent autophagy and secretion particularly in DA neurons in culture and in vivo.
Our strategy is based on the strong synergy between our teams combining biochemistry, biophysics, cell biology, electrophysiology and mouse genetics in 3 workpackages.
WP-1: we will biochemically characterize the physical and functional interaction between VAMP7 and LRRK1, LRRK2 and a-synuclein in vitro using binding assays and artificial membrane based assays. We will focus on potential phosphorylation dependent activity in the VAMP7:LRRK complexes.
WP-2: we will determine regulation by VAMP7, LRRK2 and a-synuclein of autophagosome and neuromelanin granule biogenesis, and exocytosis in PC12 cells and DA neurons. We will set up cell models for VAMP7, LRRK2, a-synuclein using CRISPR and viral vector technology and determine the role of these proteins in autophagosome biogenesis. We will characterize the biogenesis of neuromelanin granules, the secretome, and track the dynamics of VAMP7 exocytosis. We will test the effect of autophagy inducers and LRRK2 inhibitors.
WP-3: we aim to determine secretory defects in DA neurons of VAMP7, LRRK2 and a-synuclein mutants’ brains. We will use mesencephalic brain slices of VAMP7, LRRK2 and a-synuclein KO mice to characterize the secretome and the activity of GABA neurons which sense the release of dendritic DA. We will test the models for the expression of PD related proteins of the endosomal/lysosomal system. Finally, we will assess the therapeutic potential of the pharmacological induction of autophagy in the AAV-A-syn PD rodent model.
This project will lead to a better understanding of the role of key PD proteins in the regulation of vesicular trafficking. It will enable to identify specific therapeutic targets and potential biomarkers of PD related to vesicular physiology.