Synapses are fundamental to nervous system function and information processing in the normal and pathological brain. They are highly dynamic structures capable of supporting high firing rates and displaying a broad range of plasticity. A detailed picture of the molecular interactions occurring within a synapse is required to understand how synaptic protein dynamics ultimately shape activity in the nervous system in health and disease. In this proposal, we will investigate Munc13 and complexin (CPX), both highly conserved molecules that are crucial for proper synaptic function. Mice lacking complexin die at birth, and loss of a single isoform is associated with profound locomotor, sensory, and behavioral deficits. Complexin expression is altered in a host of psychiatric and neurodegenerative diseases including Huntington's, Parkinson's, and Alzheimer's disease. Although it is well-established that complexin plays a major role in synaptic function, its mode of action is controversial: there is evidence for both facilitatory and inhibitory roles of this proten in the regulation of synaptic transmission. Munc13 is a large synaptic hub protein that coordinates several proteins involved in synaptic vesicle fusion. Little is known about how neuromodulatory pathways control complexin and Munc13 function at the synapse. We propose to study the regulation of complexin and Munc13 action at the synapse in C. elegans using a combination of biochemistry, electrophysiology, genetics, and in vivo dynamic imaging. We have developed innovative methods of investigating protein interactions in vivo by monitoring the dynamics of proteins exchanging between neighboring synapses using photoactivatable GFP. We will deploy these methods in C. elegans as a model system in which to study the protein interactions of complexin and its binding partners in a functional synapse. By mutating either complexin or its binding partners, we have established that affinity changes lead to mobility changes. Similar approaches can be used to examine Munc13 interactions within individual synaptic boutons. We will also elucidate the structure of complexin and Munc13 protein domains that mediate critical membrane interactions using a combination of biochemical and spectroscopic techniques. The experiments proposed here will provide new insights into the connection between G protein-coupled receptor modulatory pathways and the inhibition of fusion by complexin. Moreover, new regulatory roles for Munc13 and CPX as well as their functional and biochemical connections will be explored.

Public Health Relevance

Synaptic connections are the fundamental building blocks of the brain. Many psychiatric and neurodegenerative diseases involve pathological synaptic function at the cellular and molecular level, but little is known about how synaptic molecules operate in health and in disease. Using a powerful combination of genetics, live animal imaging, and physiology, we propose to study complexin and Munc13, two essential proteins required for proper synapse function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095674-08
Application #
9398131
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Smith, Ward
Project Start
2011-01-01
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
8
Fiscal Year
2018
Total Cost
Indirect Cost
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
Wragg, Rachel T; Parisotto, Daniel A; Li, Zhenlong et al. (2017) Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins. Front Mol Neurosci 10:146
Michelassi, Francesco; Liu, Haowen; Hu, Zhitao et al. (2017) A C1-C2 Module in Munc13 Inhibits Calcium-Dependent Neurotransmitter Release. Neuron 95:577-590.e5
Dittman, Jeremy S; Menon, Anant K (2017) Speed Limits for Nonvesicular Intracellular Sterol Transport. Trends Biochem Sci 42:90-97
Snead, David; Lai, Alex L; Wragg, Rachel T et al. (2017) Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function. Front Mol Neurosci 10:154
Lipstein, Noa; Verhoeven-Duif, Nanda M; Michelassi, Francesco E et al. (2017) Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder. J Clin Invest 127:1005-1018
Ploier, Birgit; Caro, Lydia N; Morizumi, Takefumi et al. (2016) Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants. Nat Commun 7:12832
Wragg, Rachel T; Gouzer, GĂ©raldine; Bai, Jihong et al. (2015) Synaptic activity regulates the abundance and binding of complexin. Biophys J 108:1318-29
Snead, David; Wragg, Rachel T; Dittman, Jeremy S et al. (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955
Radoff, Daniel T; Dong, Yongming; Snead, David et al. (2014) The accessory helix of complexin functions by stabilizing central helix secondary structure. Elife 3:
Goren, Michael A; Morizumi, Takefumi; Menon, Indu et al. (2014) Constitutive phospholipid scramblase activity of a G protein-coupled receptor. Nat Commun 5:5115

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