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