The goal of this study is to characterize how neuronal synapses communicate using the Drosophila glutamatergic neuromuscular junction as a model system. Neurotransmitter release following synaptic vesicle (SV) fusion at active zones is the fundamental mechanism for presynaptic communication at synapses. Evoked release is characterized by a synchronous phase of SV fusion that occurs within milliseconds, and a slower asynchronous component that can last for hundreds of milliseconds depending on the neuronal population and firing pattern.
In Aim 1, we propose to define how the Synaptotagmin (Syt) Ca2+ sensor family regulates SV fusion. Although many molecular players involved in neurotransmitter release have been identified, how they work mechanistically to control fusion is still unclear. Genetic analysis of Syt 1 has confirmed that it is responsible for sensing Ca2+ influx and driving synchronous fusion of SVs, while also suppressing asynchronous release. In addition to Syt 1, the Syt 7 isoform has been suggested to function as the asynchronous Ca2+ sensor, although this model is still controversial. We will use a structure-function approach to characterize how these two Ca2+ sensors regulate SV fusion. Using a large collection of new point mutants we have generated in Syt 1, we will test how Syt dimerization, docking onto the plasma membrane, and interactions with Complexin and the SNARE complex regulate SV trafficking and fusion. Using Syt 7 and Syt 1/Syt 7 double mutants we will also determine if Syt 7 functions as the asynchronous Ca2+ sensor, or may instead regulate SV availability during high frequency firing. In contrast to SV trafficking, we know little about the regulation of postsynapti vesicle fusion and how retrograde signals modulate synapse biology.
In Aim 2, we will characterize the role of the postsynaptic Syt isoform, Syt 4, in regulating Ca2+-regulated vesicle trafficking that underlies retrograde signaling. We have identified Syntaxin 4 as the essential t-SNARE for postsynaptic exocytosis and have shown that the two proteins regulate acute changes in both synaptic structure and function. We will employ APEX labeling techniques and genetic analysis of Syt 4-pHlourin trafficking to define and characterize the postsynaptic vesicle proteome, allowing us to develop a more complete picture of postsynaptic vesicle trafficking and how it contributes to synaptic signaling. The proposed studies will provide important insights into how synapses use vesicular trafficking pathways to mediate bi-directional synaptic communication, providing a foundation to understand how neurological and psychiatric diseases may disrupt these essential elements of neuronal signaling.

Public Health Relevance

The proposed research will define basic mechanisms that mediate neurotransmitter release and retrograde signaling at neuronal synapses. Alterations in synaptic communication have been linked to numerous neurological and psychiatric diseases of the human brain. By defining how synapses bi-directionally communicate, our research will provide a foundation for understanding brain diseases that alter the ability of the synapse to properly signal.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Synapses, Cytoskeleton and Trafficking Study Section (SYN)
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Talley, Edmund M
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Harris, Kathryn P; Littleton, J Troy; Stewart, Bryan A (2018) Postsynaptic Syntaxin 4 negatively regulates the efficiency of neurotransmitter release. J Neurogenet 32:221-229
Zhang, Yao V; Ormerod, Kiel G; Littleton, J Troy (2017) Astrocyte Ca2+ Influx Negatively Regulates Neuronal Activity. eNeuro 4:
Guan, Zhuo; Bykhovskaia, Maria; Jorquera, Ramon A et al. (2017) A synaptotagmin suppressor screen indicates SNARE binding controls the timing and Ca2+ cooperativity of vesicle fusion. Elife 6:
Akbergenova, Yulia; Littleton, J Troy (2017) Pathogenic Huntington Alters BMP Signaling and Synaptic Growth through Local Disruptions of Endosomal Compartments. J Neurosci 37:3425-3439
Krench, Megan; Cho, Richard W; Littleton, J Troy (2016) A Drosophila model of Huntington disease-like 2 exhibits nuclear toxicity and distinct pathogenic mechanisms from Huntington disease. Hum Mol Genet 25:3164-3177
Weiss, Kurt R; Littleton, J Troy (2016) Characterization of axonal transport defects in Drosophila Huntingtin mutants. J Neurogenet 30:212-221
Harris, Kathryn P; Zhang, Yao V; Piccioli, Zachary D et al. (2016) The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling. Elife 5:
Cho, Richard W; Buhl, Lauren K; Volfson, Dina et al. (2015) Phosphorylation of Complexin by PKA Regulates Activity-Dependent Spontaneous Neurotransmitter Release and Structural Synaptic Plasticity. Neuron 88:749-61
Whittaker, Roger G; Herrmann, David N; Bansagi, Boglarka et al. (2015) Electrophysiologic features of SYT2 mutations causing a treatable neuromuscular syndrome. Neurology 85:1964-71
Lee, Jihye; Littleton, J Troy (2015) Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release. Proc Natl Acad Sci U S A 112:3793-8

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