Role of Endoplasmic Reticulum Inter-Organelle Contacts in Neuronal Endosomal Traffic - This proposal aims to determine how the endoplasmic reticulum (ER) is regulated in neurons at rest and during neuronal activity. The ER is a contiguous membrane-bound compartment, which forms a network of tubules that extends to the periphery of neurons and forms contacts with other organelles, controlling processes such as lipid transfer, organelle scission, and calcium signaling. Patients with neurodegenerative disorders, such as Hereditary Spastic Paraplegia (HSP), often have mutations in genes that disrupt ER structure and ER contacts. However, our understanding of ER function in neurons primarily comes from studies of non-neuronal cells. The complex morphologies and elongated structures of neurons impose unique challenges for secretory and endosomal membrane trafficking pathways. In particular, endosomes are vital for conveying long-distance signals within neurons and packaging cargoes for intercellular signaling through extracellular vesicles. We currently know very little about how ER contacts regulate neuronal endosomal compartments, or how the structure of the neuronal ER is modified by activity. This project will use Drosophila and mammalian neuronal systems to examine ER-endosome contacts in neurons and to study how neuronal activity regulates the ER. The Drosophila neuromuscular junction is an excellent system to study the ER because it is genetically tractable, and easily accessible for live imaging and electrophysiology. On the other hand, mammalian neuronal cultures provide homogeneous material for biochemical studies, and can show if findings in Drosophila are evolutionarily conserved. First, this proposal will determine how the HSP-linked gene Atlastin, which controls the assembly of the ER into a reticular network, affects ER-endosome contacts in Drosophila and mammalian neurons (Aim1a,c). Atlastin mutant synapses will be examined to uncover the mechanistic basis of the observed structural and dynamic defects in the ER, as well as the accumulation of endosomal and extracellular vesicle cargoes (Aim1b). Next, this study will examine how different neuronal activity paradigms (e.g. spontaneous vs. mild vs strong stimulation) regulate the dynamics of the ER and ER-endosome contacts in Drosophila (Aim2a). Finally, proximity labeling approaches will also be used in hippocampal neurons to identify candidate proteins at ER contacts that are regulated by neuronal activity, to lay the foundation for future functional studies in Drosophila (Aim2b). To achieve these research goals, the proposed training plan will expand the PI's experimental skillsets in various microscopy techniques, construction of genetic sensors, mammalian neuronal culture, and new coding skills for image analysis such as image restoration and particle tracking. The career development plan focuses on strengthening the PI's publication record and transitioning to an independent research position. Overall, the K99/R00 will allow the PI to gain necessary skills to become an independent researcher in the field of neuronal cell biology and membrane trafficking.
