Research Project
10474868
PROJECT SUMMARY ?-Synuclein (?-Syn) is a protein abundantly distributed in presynaptic terminals. ?-Syn pathology is linked to synucleinopathies including Parkinson?s disease (PD) and Lewy body dementia, and ?-Syn missense muta- tions cause early onset PD. Physiologically, ?-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 ?-Syn simultaneously decrease with increasing duration of dementia, which implies a potential functional link between VAMP2 and normal monomeric ?-Syn. Preliminary data showed VAMP2 interacts with ?-Syn to prevent ?-Syn aggregation. The long-term goal of this project is to define and characterize critical protein factors that stabilize binding of ?-Syn on SVs and test their effect on neuron function and ?-Syn induced toxicity. The central hy- pothesis is that VAMP2 stabilizes ?-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 ?-Syn aggregation and toxicity; 2) Determine ?-Syn?s function in SNARE-mediated fusion in vitro; 3) Determine the influence of VAMP2 on ?-Syn?s function and toxicity in vivo. Under the first aim, ?-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 ?-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 ?-Syn variants. Under the third aim, localization, function, and aggregation of ?-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 ?-Syn with increased SV binding. This research is significant because it will (1) clarify the importance of SV binding of ?-Syn for SNARE-complex assembly and neuron function, (2) provide novel insights into the function/dysfunction of recently identified PD-mutations of ?- Syn, (3) provide new insights into the molecular mechanism underlying SV-binding of ?-Syn by altering VAMP2 levels, and has (4) translational importance for the targeted development of new strategies aimed at preserving ?-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 ?-Syn function and early oligomerization, and (3) tests the novel hypothesis that VAMP2 is important stabilizers of SV-binding of ?-Syn and that stabili- zation of the SV-bound pool of ?-Syn prevents its aggregation and toxicity.
