Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. Synapses are made up of crucial cellular machineries that orchestrate a balance of membrane traffic to and from the plasma membrane. Our goal is to develop detailed quantitative understanding of the synapse both in terms of physiological responses to action potential stimuli as well as the molecular underpinnings of its function. We recently developed sensitive approaches that allow us to determine the detailed kinetics of endocytosis as well as the relative size of recycling and resting vesicle pools. These methods allow us to perform functional tests of how specific proteins are controlling various aspects of presynaptic function. The goal of this project has 2 main AIMS.
Our first AIM will extend our recent findings that the recycling and resting vesicle pool fractions in nerve terminals is controlled by the balance of two enzymatic activities, the phosphatase Calcineurin and the kinase CDK5. Our goal is to specifically test the hypothesis that the abundant presynaptic phosphoproteins Synapsin and -catenin which are substrates of CDK5 are critical to mediating this control of these vesicle pools. We will also determine which cyclin controls CDK5.
The second AIM will explore a fundamental property of nerve terminals, the retrieval of synaptic vesicle components. We will test the hypothesis that during vesicle recycling the different SV membrane proteins remain together, by simultaneously observing the endocytic behavior of different SV proteins. We will examine the importance of the known change in phosphorylation state (driven by CDK5) that dynamins, amphiphysin and synaptojanin undergo with activity by examining endocytosis properties at nerve terminals in which these proteins have been fully replaced by phosphomimetic mutants. Finally in our recent work we discovered that even in the absence of major brain dynamins 1 &3 endocytosis persists. This result is enormously surprising and we are compelled to understand how endocytosis can be achieved in the absence of these once-thought critical mechanoenzymes. We will test hypotheses that the endocytosis that persists in the absence of the major brain dynamins, Dynamin 1 and 3 is driven by either the remaining dynamin (Dynamin 2) or one of two known BAR domain proteins, Amphiphysin or Syndapin.

Public Health Relevance

Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. The functioning of the human brain relies on the careful orchestration of delivering neurotransmitter-laden vesicles to sites at nerve terminals where they can be used to deliver this chemical message on demand. Many known genetic mutations in diseases such as Parkinson's disease, migraine headache and schizophrenia are linked to proteins that control synapse function. Our work is aimed at understanding the machinery at a molecular level to better ensure the success of future therapies for these types of neuronal diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS036942-15
Application #
8269871
Study Section
Special Emphasis Panel (ZRG1-MDCN-N (02))
Program Officer
Talley, Edmund M
Project Start
1997-12-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
15
Fiscal Year
2012
Total Cost
$369,688
Indirect Cost
$150,938
Name
Weill Medical College of Cornell University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Rangaraju, Vidhya; Calloway, Nathaniel; Ryan, Timothy A (2014) Activity-driven local ATP synthesis is required for synaptic function. Cell 156:825-35
Kim, Sung Hyun; Ryan, Timothy A (2013) Balance of calcineurin A? and CDK5 activities sets release probability at nerve terminals. J Neurosci 33:8937-50
Ariel, Pablo; Ryan, Timothy A (2012) New insights into molecular players involved in neurotransmitter release. Physiology (Bethesda) 27:15-24
Armbruster, Moritz; Ryan, Timothy A (2011) Synaptic vesicle retrieval time is a cell-wide rather than individual-synapse property. Nat Neurosci 14:824-6
Raimondi, Andrea; Ferguson, Shawn M; Lou, Xuelin et al. (2011) Overlapping role of dynamin isoforms in synaptic vesicle endocytosis. Neuron 70:1100-14
Kim, Sung Hyun; Ryan, Timothy A (2010) CDK5 serves as a major control point in neurotransmitter release. Neuron 67:797-809
Kim, Sung Hyun; Ryan, Timothy A (2009) Synaptic vesicle recycling at CNS snapses without AP-2. J Neurosci 29:3865-74
Fernandez-Alfonso, Tomas; Ryan, Timothy A (2008) A heterogeneous "resting" pool of synaptic vesicles that is dynamically interchanged across boutons in mammalian CNS synapses. Brain Cell Biol 36:87-100
Balaji, J; Armbruster, Moritz; Ryan, Timothy A (2008) Calcium control of endocytic capacity at a CNS synapse. J Neurosci 28:6742-9
Ferguson, Shawn M; Brasnjo, Gabor; Hayashi, Mitsuko et al. (2007) A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis. Science 316:570-4

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