Investigation of SYD-2 as a Neuronal Substrate of the Anaphase-promoting Complex Enzyme in C. elegans

Neurons communicate at specialized cell junctions called synapses by releasing chemical neurotransmitters (NTs) that bind to postsynaptic neurons, causing either inhibitory or excitatory responses. Proteins in pre- and post-synaptic neurons work to regulate synaptic transmission. One regulator of synaptic signaling is ubiquitin (Ub), a polypeptide that causes protein degradation. The ubiquitin-proteasome system (UPS) degrades most proteins, and a failure to properly degrade proteins occurs in many neurodegenerative diseases. The Kowalski lab showed the anaphase-promoting complex (APC), an enzyme that adds Ub to proteins, regulates inhibitory synaptic signaling at neuromuscular synapses in C. elegans roundworms. Potential APC substrates at this synapse include SYD-2, a synaptic scaffolding protein, and we found synaptic SYD-2 levels increase in APC loss of function mutants. However, it is unclear if the APC or proteasome directly control SYD-2 ubiquitination. First, if SYD-2 is a substrate of the APC at the C. elegans NMJ, then Ub-SYD-2 levels should fall and overall SYD-2 abundance increase in APC mutants compared to wildtype worms. Thus far, a Ub-SYD-2 band has been detected, but additional tests are needed to determine if the band is specific and how it compares to Ub-SYD-2 levels in wildtype animals. Second, if the proteasome regulates SYD-2, then total SYD-2 and Ub-SYD-2 levels should increase with proteasome inhibition. Preliminary results have shown no apparent difference in SYD-2 levels at the inhibitor concentrations or exposure times tested. Results of additional trouble-shooting will be presented. These studies are important in determining if SYD-2 is a potential substrate for the APC. Biolog