The role of VAMP2 in alpha-synuclein function and pathology

PROJECT SUMMARY α-Synuclein (α-Syn) is a protein abundantly distributed in presynaptic terminals. α-Syn pathology is linked to synucleinopathies including Parkinson’s disease (PD) and Lewy body dementia, and α-Syn missense muta- tions cause early onset PD. Physiologically, α-Syn functions in synaptic transmission via clustering synaptic vesicles (SVs) and promoting SNARE-complex assembly. This function strongly depends on binding to SVs. Pathologically, levels of vesicle-associated membrane protein 2 (VAMP2) and functional monomeric α-Syn simultaneously decrease with increasing duration of dementia, which implies a potential functional link between VAMP2 and normal monomeric α-Syn. Preliminary data showed VAMP2 interacts with α-Syn to prevent α-Syn aggregation. The long-term goal of this project is to define and characterize critical protein factors that stabilize binding of α-Syn on SVs and test their effect on neuron function and α-Syn induced toxicity. The central hy- pothesis is that VAMP2 stabilizes α-Syn in its functional conformation on SVs to mediate vesicle clustering and SNARE-complex assembly, and to prevent pathological aggregation. Guided by strong preliminary data, this hypothesis will be tested in three specific aims: 1) Determine how VAMP2 prevents α-Syn aggregation and toxicity; 2) Determine α-Syn’s function in SNARE-mediated fusion in vitro; 3) Determine the influence of VAMP2 on α-Syn’s function and toxicity in vivo. Under the first aim, α-Syn interaction with VAMP2, folding and oligomerization will be analyzed in solution and on membranes, using NMR, ThioflavinT, CD, FRET and solid- state nanopore experiments on recombinant α-Syn variants. Under the second aim, the mechanism and kinet- ics of SNARE-mediated membrane fusion will be analyzed, using single-molecule fusion, vesicle clustering and NMR experiments on recombinant α-Syn variants. Under the third aim, localization, function, and aggregation of α-Syn variants will be analyzed, using biochemical and cell biological assays on primary mouse neurons, in addition to behavioral and pathology readouts in wild-type and heterozygous VAMP2 mice. The study is ex- pected to show improved function and reduced toxicity of α-Syn with increased SV binding. This research is significant because it will (1) clarify the importance of SV binding of α-Syn for SNARE-complex assembly and neuron function, (2) provide novel insights into the function/dysfunction of recently identified PD-mutations of α- Syn, (3) provide new insights into the molecular mechanism underlying SV-binding of α-Syn by altering VAMP2 levels, and has (4) translational importance for the targeted development of new strategies aimed at preserving α-Syn’s function via stabilizing its SV-bound pool through elevating VAMP2 levels. Our study is innovative be- cause it (1) uses a multidisciplinary approach combining biophysical, biochemical, and cell biological ap- proaches, (2) uses unique single-particle detection assays to study α-Syn function and early oligomerization, and (3) tests the novel hypothesis that VAMP2 is important stabilizers of SV-binding of α-Syn and that stabili- zation of the SV-bound pool of α-Syn prevents its aggregation and toxicity.