The ubiquitin signaling system, which promotes regulated protein degradation, is essential for synapse development and synaptic transmission. Hundreds of enzymes exist that have the ability to add or remove ubiquitin polypeptides from proteins. Less is known about the functions and relevant substrates of these enzymes in neurons and at synapses. The long-term goal is to determine how ubiquitin enzymes regulate synaptic transmission. Given that ubiquitin system dysfunction is linked to neurological and neurodegenerative diseases in which aberrant synaptic transmission occurs, such information may lead to the identification of drug targets to aid in treating these conditions. Recent work established that the Anaphase Promoting Complex (APC), an ubiquitin ligase that controls protein abundance in the cell cycle, also regulates neuronal development and glutamatergic synaptic transmission;yet much remains unknown regarding its mechanism of action and relevant substrates at these or other synapses. The objective of this proposal is to elucidate the molecular mechanisms by which the APC regulates synaptic transmission in vivo. This will be done using the model nematode, Caenorhabditis elegans, which shares significant conservation of nervous system proteins and function with humans. Preliminary experiments indicate that, in addition to regulating glutamatergic synapses, the APC negatively regulates transmission at the neuromuscular junction (NMJ), a tripartite synapse in C. elegans where contraction of postsynaptic muscle is controlled by a balance of inputs from excitatory cholinergic and inhibitory GABAergic motorneurons. The APC may act in any or all of these cell types to impact the amount of muscle activity. Additional preliminary localization studies and behavioral assays led to the central hypothesis that the APC acts in presynaptic motorneurons to promote ubiquitination and degradation of NMJ proteins important for increasing muscle excitation.
In Aim 1, cell type-specific loss of function and rescue experiments, behavioral assays, and quantitative imaging will be used to determine the site and mechanism of APC action during NMJ signaling. Additional preliminary data led to the identification of one potential APC substrate at the NMJ, the G protein-coupled receptor, FSHR-1.
In Aim 2, genetic and behavioral analyses, along with imaging and biochemistry, will be employed to characterize the role of FSHR-1 at the NMJ and the relationship between FSHR-1 and the APC.
In Aim 3, RNA interference will be used to identify additional APC substrates. This research will provide new insight into how the balance of excitatory to inhibitory transmission is regulated-a balance lost i human seizure syndromes, such as epilepsy. These studies also will contribute to the global picture of how ubiquitin signaling impacts synaptic transmission, which is critical for learning an memory. Given that ubiquitin system misregulation occurs in Alzheimer's, Parkinson's, and Huntington's Diseases, and mutations in ubiquitin enzymes are linked to several neurogenetic disorders, elucidating how the APC and its substrates control synaptic transmission may facilitate the development of relevant therapeutics.

Public Health Relevance

This project involves determining the molecular mechanism by which the Anaphase Promoting Complex (APC), an ubiquitin ligase enzyme, regulates synaptic transmission in vivo. Proper signaling at neuronal synapses is required for learning and memory and underlies all nervous system function. Misregulation of synaptic transmission and ubiquitin system dysfunction occur in many neurological disorders. Thus, a more complete understanding of the mechanism of action and relevant substrates of the APC ubiquitin ligase will contribute critical information to the global picture of synaptic transmission and ubiquitin enzyme activity.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Academic Research Enhancement Awards (AREA) (R15)
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Special Emphasis Panel (ZRG1-MDCN-E (96))
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Talley, Edmund M
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Butler University
Schools of Arts and Sciences
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Kowalski, Jennifer R; Dube, Hitesh; Touroutine, Denis et al. (2014) The Anaphase-Promoting Complex (APC) ubiquitin ligase regulates GABA transmission at the C. elegans neuromuscular junction. Mol Cell Neurosci 58:62-75
Kowalski, Jennifer R; Juo, Peter (2012) The role of deubiquitinating enzymes in synaptic function and nervous system diseases. Neural Plast 2012:892749