Investigation of the Novel Role and Underlying Mechanism of Znf179 in Synaptic Trafficking

Endosomes are important in membrane trafficking and play a crucial role in regulating synaptic strength and plasticity by controlling recycling of presynaptic vesicles and postsynaptic receptors. Zinc finger protein 179 (Znf179) has been found to peripherally associate with the membrane and localize to endosomes to affect endosomal membrane dynamics. Previous studies, including ours, observed reduced paired-pulse facilitation and long-term potentiation in Znf179 knockout mice, suggesting pre- and postsynaptic contributions of Znf179. Our preliminary result showed that many synaptic proteins such as STXBP1 (syntaxin binding protein 1), drebrin, Rab11fip5 (RAB11 family interacting protein 5), and the kinesin superfamily proteins (KIFs) that have important pre- and postsynaptic functions such as endocytosis, exocytosis, microtubule dynamics, and synaptic transmission were identified by Znf179 IP-mass spectrometry analysis. Using co-immunoprecipitation coupled with Western blotting, we will further confirm the interaction of Znf179 and these possible interacting partners. GST pull-down and in vitro ubiquitination assays will be used to examine direct interactions between Znf179 and the possible interacting proteins and whether Znf179 can mediate their ubiquitination. Using FM dye imaging, we will examine the changes of synaptic vesicle exocytosis in wild type and Znf179 knockout neurons. We will also use biochemical fractionation coupled with Western blotting to detect the neurotransmitter receptors trafficking in wild type and Znf179 knockout mice after LTP induction. The interactions between potential Znf179 interacting proteins and their effectors will also be examined in the presence or absence of Znf179. Further investigation of the effects and underlying mechanisms of Znf179 in synaptic functions will enhance our understanding of the mechanisms of synaptic trafficking and synaptic plasticity.