In response to RFA-MH-12-140, "Development of Tools to Explore the Synaptome", we propose to develop a procedure to trap and isolate recycling synaptic vesicles as an essential first step in analyzing how vesicle protein composition varies with changes in synaptic activity, drug treatment, and under pathological conditions. The ability of synaptic vesicles to undergo multiple rounds of fusion is crucial to maintaining fast, efficient neurotransmission. Synaptic vesicles are formed and recycle via endocytosis. At many synapses, a small proportion of the total vesicle population mediates the majority of neurotransmission while the larger portion remains untapped in a reserve pool. Therefore, proteomic analyses using traditional vesicle purification schemes cannot track changes in the protein composition of the recycling pool. To address this we will develop a scalable, readily applied purification protocol for recycling synaptic vesicles that relies on transiently trapping recycling vesicles as a means of separating them from the total vesicle pool. This approach will open a new field of investigation into the complex endocytotic machinery that regulates vesicle recycling. It will provide a means to test the effects of drugs, genetic manipulations, and pathological conditions on this very important process.

Public Health Relevance

Increased understanding of the molecular events involved in cellular processes is an essential first step towards transformative therapies. Here we propose to develop a procedure to capture and isolate recycling synaptic vesicles. This procedure will allow scientists to determine how synaptic activity alters synaptic vesicle composition and to identify the proteins involved in sorting molecules to the vesicle under normal and pathological conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21MH099741-02
Application #
8585105
Study Section
Special Emphasis Panel (ZMH1-ERB-S (05))
Program Officer
Nadler, Laurie S
Project Start
2012-12-01
Project End
2014-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
2
Fiscal Year
2014
Total Cost
$173,813
Indirect Cost
$61,313
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195