The computational power of the brain depends on synaptic connections that link together billions of neurons. With the long-term goal of understanding how synaptic signaling regulates neuronal communication and connectivity, and how its dysfunction contributes to neurological disease, we propose to use Drosophila as a model system for determining the molecular mechanisms underlying neurotransmitter release and retrograde signaling. Despite the differences in complexity between flies and mammals, genomic analysis suggests many key neuronal proteins and the functional mechanisms they govern are remarkably similar.
In Aim 1, we propose to elucidate the underlying molecular interactions mediated by the SNARE binding proteins Synaptotagmin 1 and Complexin that mediate rapid information transfer from the presynaptic terminal. We have generated screening approaches that allow F1 genetic screens for Synaptotagmin 1 point mutants, allowing us to do saturation mutagenesis to identify key residues and protein interactions that are required for the regulation of synaptic vesicle fusion. In addition we will determine how the Ca2+ sensor Synaptotagmin 1 and the synaptic vesicle fusion clamp Complexin coordinately regulate information transfer from the presynaptic terminal. These studies will generate new mechanistic insights into the synaptic vesicle fusion machinery that will go well beyond our current understanding of neurotransmitter release.
In Aim 2, we propose to define the molecular machinery for Ca2+-regulated postsynaptic release that mediates retrograde signaling. Unlike the synaptic vesicle-associated Synaptotagmin 1, we found that Synaptotagmin 4 is expressed postsynaptically and mutants lacking Synaptotagmin 4 show abnormal development and function of synapses. Our genetic analysis of synaptic plasticity in Drosophila suggests that Ca2+-dependent retrograde vesicular trafficking mediated by Synaptotagmin 4 initiates an acute change in synaptic function that is converted to synapse- specific growth, providing a link between synaptic plasticity and activity-dependent rewiring of neuronal connections. We will characterize this retrograde signaling pathway by identifying additional components of the postsynaptic vesicle fusion machinery and the retrograde signals involved. We will also compare the functional properties of the two Synaptotagmin isoforms in mediating pre- versus post-synaptic vesicle fusion. The proposed studies will provide important insights into how the nervous system functions at the cellular level, allowing us to integrate this information into the framework of ultimately understanding how neuronal ensembles mediate behavior, and how neurological and psychiatric diseases disrupt these processes.

Public Health Relevance

This research will define basic mechanisms underlying neurotransmitter release and retrograde signaling during synaptic plasticity. Alterations in synaptic signaling have been linked to numerous neurological and psychiatric diseases of the human brain. By defining the mechanisms of synaptic communication, our research will provide a foundation for developing potential therapeutic approaches for brain diseases that alter the ability of the synapse to signal properly.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS040296-12
Application #
8467060
Study Section
Special Emphasis Panel (ZRG1-MDCN-F (05))
Program Officer
Talley, Edmund M
Project Start
2000-07-01
Project End
2015-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
12
Fiscal Year
2013
Total Cost
$353,582
Indirect Cost
$142,488
Name
Massachusetts Institute of Technology
Department
Miscellaneous
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Herrmann, David N; Horvath, Rita; Sowden, Janet E et al. (2014) Synaptotagmin 2 mutations cause an autosomal-dominant form of lambert-eaton myasthenic syndrome and nonprogressive motor neuropathy. Am J Hum Genet 95:332-9
Cho, Richard W; K├╝mmel, Daniel; Li, Feng et al. (2014) Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo. Proc Natl Acad Sci U S A 111:10317-22
Piccioli, Zachary D; Littleton, J Troy (2014) Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM kinase regulation of cofilin. J Neurosci 34:4371-81
Blunk, Aline D; Akbergenova, Yulia; Cho, Richard W et al. (2014) Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction. Mol Cell Neurosci 61:241-54
Lee, Jihye; Guan, Zhuo; Akbergenova, Yulia et al. (2013) Genetic analysis of synaptotagmin C2 domain specificity in regulating spontaneous and evoked neurotransmitter release. J Neurosci 33:187-200
Buhl, Lauren K; Jorquera, Ramon A; Akbergenova, Yulia et al. (2013) Differential regulation of evoked and spontaneous neurotransmitter release by C-terminal modifications of complexin. Mol Cell Neurosci 52:161-72
Melom, Jan E; Akbergenova, Yulia; Gavornik, Jeffrey P et al. (2013) Spontaneous and evoked release are independently regulated at individual active zones. J Neurosci 33:17253-63
Yoshihara, Motojiro; Guan, Zhuo; Littleton, J Troy (2010) Differential regulation of synchronous versus asynchronous neurotransmitter release by the C2 domains of synaptotagmin 1. Proc Natl Acad Sci U S A 107:14869-74
Cho, Richard W; Song, Yun; Littleton, J Troy (2010) Comparative analysis of Drosophila and mammalian complexins as fusion clamps and facilitators of neurotransmitter release. Mol Cell Neurosci 45:389-97
Wu, Chaohong; Schulte, Joost; Sepp, Katharine J et al. (2010) Automatic robust neurite detection and morphological analysis of neuronal cell cultures in high-content screening. Neuroinformatics 8:83-100

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