The Role of VAP Membrane Contact Sites in Axonal Calcium Dynamics and Synaptic Transmission - Project Summary The neuronal endoplasmic reticulum (ER) extends throughout the cytoplasm and forms extensive physical contacts with organelles, even within specialized compartments like the presynaptic terminal. These contact sites between the ER and other organelles are termed membrane contact sites (MCSs). MCSs are held together by tethering proteins and are proposed to contribute to at least three cellular functions: trafficking of lipids, organelle positioning, and exchange of calcium (Ca2+) ions. During action potential (AP) signaling, the influx of Ca2+ through voltage-gated Ca2+ channels forms localized Ca2+ microdomains. These Ca2+ microdomains are critical for synaptic vesicle fusion, signaling through Ca2+-sensitive proteins, and organelle function. MCSs are thought to contribute to Ca2+ exchange by recruiting ER-bound proteins and organelles to Ca2+ microdomains, although it is unclear which organelles are most reliant on tethering to the ER. A fundamental gap exists in understanding the molecular composition and function of tethering proteins in the axon. The development of optogenetic Ca2+ indicators of different affinities has provided unprecedented opportunities to study how MCSs in the axon link electrical activity with Ca2+ signaling to support synaptic function. VAMP-Associated Protein (VAP) is a highly conserved ER tethering protein that binds numerous intracellular targets. In the axon, VAP forms a unique MCSs by binding to voltage-gated potassium (Kv) channels Kv2 at the plasma membrane (PM). Disrupting these ER/PM MCSs by knocking down Kv2 channels impairs synaptic vesicle exocytosis by ~50% and requires VAP interactions in the Kv2 C-terminus. Unexpectedly when VAP is knocked down in excitatory neurons, there is no change in AP-evoked cytosolic Ca2+ despite a ~50% impairment in exocytosis. The central hypothesis is that Kv2/VAP MCSs are an essential structural component of presynaptic terminals for Ca2+ signaling microdomains to support synaptic function. The overall objective is to determine at what level membrane tethering affects synaptic function. The long-term goals are to determine how membrane tethering informs synaptic strength and how dysregulation of these processes are involved in disease. Aim 1 will determine how VAP domains contribute to synaptic function using optical indicators of synaptic vesicle exocytosis. Aim 2 will determine how VAP MCSs influence Ca2+ dynamics within axonal organelles using subcellular optical indicators of Ca2+. Given the complexity of this system and the essential nature of electrical signaling in excitable cells, it is unsurprising that mutations in VAPB are implicated in several diseases including familial amyotrophic lateral sclerosis. There is a critical need for a better understanding of the role of membrane tethering in intracellular Ca2+ dynamics in the axon that we know is critical for synaptic function.
