The endocytosis and recycling of synaptic vesicles in nerve terminals is essential to sustain neurotransmission. PLD1 and 2 and their enzymatic product PA, have been shown to be important regulators in these processes. The goal of this proposal is to better understand the role of PA, and PLD- derived PA, in particular, in endocytosis and vesicular trafficking. Insights into plasma membrane (PM) PA function will be gained by disrupting the levels of this lipid at the PM. We resort to achieving this using two approaches. The first employs localized constitutive expression of PA metabolizing enzymes that either up- or downregulate PA at the PM using a targeting vector. The second uses an acute recruitment device that subjects PM recruitment of these enzymes to a pharmacological trigger. The functional effects of PM PA modulation on endocytosis and recycling will be tested in various biochemical assays and by fluorescent microscopy. The PLD pathway is the predominant pathway for the synthesis of for pools of PA that are relevant for endocytosis and vesicular trafficking. Specifically, PLD1 and 2 appear to have roles in regulated secretion and receptor mediated endocytosis respectively, but, to date, there is no genetic evidence in support of this data. Still, there remains the possibility functional synergy and/or redundancy between the two isoforms. Thus, our lab has generated both PLD1 and PLD2 KO mice, and experiments are underway to generate the PLD1/2 KO mice. Collectively, these tools allow us to address the role of PLD in endocytosis and recycling and more specifically, the respective contributions of each of the isoforms in these processes. First, mice will be used as a source of both primary and immortalized fibroblasts in a variety of endocitic and recycling assays. Mice will also be used as a source of hippocampal neurons for conducting dye - uptake/release assays along with experiments utilizing a relatively new endocytic rate-indicator tool, synaptopHluorin. Finally, our study will also involve rescue experiments with exogenous mutants of the PLD gene in these mutant backgrounds in an effort to further finely dissect the mechanistic details of PLD contribution in these processes. We anticipate that our studies will provide insight into the role of PA and PLD in endocytosis and vesicular trafficking. Understanding the molecular machinery that drives endocytosis and vesicular trafficking is critical to our understanding of neurotransmission and will give insight into related mechanistic processes that are aberrant in diseases such as Alzheimer's and Parkinson's disease for which links to PLD have been discovered.
