Dynamic Regulation of Vesicular Trafficking by Altered Sterol Homeostasis

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PROJECT SUMMARY A fundamental cellular process critical for normal neurodevelopment and neuronal function is clathrin-mediated endocytosis. Within the CNS, clathrin-mediated endocytosis is functionally coupled to the exocytosis of synaptic vesicles for neurotransmission and essential for vesicular receptor desensitization. While cholesterol depletion is known to dramatically inhibit clathrin-mediated signaling, the specific mechanisms and requirements underlying membrane dynamics and clathrin-mediated endocytic pathways are unknown. Interestingly, autosomal recessive disorders of cholesterol synthesis, characterized by substitution of cellular cholesterol for sterol intermediates, constitute a group of malformation syndromes that severely affect nervous system development and function. Our preliminary data demonstrates clathrin-mediated endocytosis exhibits a high degree of lipid specificity to function normally. Delineating the neurological consequences of altered sterol homeostasis on clathrin activity will provide novel mechanistic data regarding vesicular trafficking in the context of neurodevelopment and neuronal function. Our long-term objective for this proposal is to delineate the cellular consequences of sterol substitution on vesicular trafficking and neuronal function. Aim 1 will utilize live- cell imaging of CRISPR-Cas9-edited human induced pluripotent stem cells (iPSCs) to define the impact of altered sterol homeostasis on clathrin-mediated endocytosis. Endogenous clathrin dynamics will be monitored in real-time and quantified by live cell confocal imaging, fluorescence recovery after photobleaching (FRAP), and total internal reflection fluorescence (TIRF) microscopy. Aim 2 will determine the molecular mechanism by which cholesterol synthesis inhibition disrupts clathrin-mediated endocytic events through atomic force microscopy and polarized total internal reflection fluorescence (polTIRF) microscopy to dynamically monitor cell stiffness, membrane bending and clathrin assembly. Aim 3 will define the functional effects of sterol substitution on vesicular trafficking and clathrin-mediated endocytosis within the neural synapse using patch- clamp electrophysiology and high-resolution microscopic analysis of differentiated patient-derived iPSCs. These studies will directly contribute to the understanding of clathrin-mediated endocytosis and functional implications of disruption of this process by sterol precursors on vesicular trafficking at the mammalian synapse. This work will provide critical evidence for disrupted clathrin-mediated trafficking underlying the neurological deficits observed in cholesterol synthesis disorders and may support a role for dysfunction of clathrin-mediated endocytosis in more common neurological conditions associated with altered cholesterol levels, such as schizophrenia, Huntington's disease, and Alzheimer's disease.