The Role of Non-Vesicular Lipid Transport at ER-PM Contact Sites in Phosphoinositide Signaling in Early Dendrite Development - Abstract Phosphoinositide lipid signaling is implicated in many cellular functions and is crucial in embryonic neuronal development. PI(4,5)P2 is a key lipid at the plasma membrane (PM), mediating various signaling pathways downstream of secreted cues, directly downstream of PI(4,5)P2 or following its phosphorylation to PI(3,4,5)P3 (PIP3) by PI3-Kinase. PIP3 and Akt signaling are critically implicated in dendrite development, effects largely mediated via diffusible neurotrophins. Neurotrophins BDNF and NT-3 expression are elevated in dendrites during and play a critical role in dendrite growth and branching. PI(4,5)P2 homeostasis during receptor stimulation is maintained by a cyclical metabolic pathway, phospha- tidylinositol (PI) cycle. The PI cycle and the enzymatic machinery required to sustain it are spatially segregated between the ER and PM. In the developing brain, neurons are perpetually exposed to cues that trigger the use and hydrolysis of PM PI(4,5)P2. Developing neurons must accommodate rapid on-demand PI availability at targeted locations of PI(4,5)P2 signaling and turnover in response to external cues, during extensive morphogenesis in dendrite development. The slow and non-specific vesicular transport might not meet this demand. Recent evidence suggests that an alternative mode of lipid transport, by lipid transport proteins at ER-PM contact sites, may provide a localized, rapid on-demand replenishment of PM PI(4,5)P2 and drive its downstream signaling following its hydrolysis in response to external cues. ER is ubiquitously present in dendrites of early developing neurons. Thus, PM PI(4,5)P2 replenishment at ER-PM contacts might be prominent during extensive morphogenesis in embryonic dendrite development. This study determines whether the lipid transport protein Nir2 is a critical regulator of PI(4,5)P2/PIP3 homeostasis at ER-PM contact sites, and in PI(4,5)P2/PIP3 signaling in dendrite development. The Phosphatidylinositol Transfer Protein (PITP), Nir2, can provide precursors of PI(4,5)P2 to PM while also extracting PI(4,5)P2 metabolite PA from PM for PI synthesis in the ER, thus autonomously sustaining the PI cycle at localized regions of PI(4,5)P2 depletion. Furthermore, Nir2 is recruited to PM only following PI(4,5)P2 hydrolysis. This makes Nir2 an optimal candidate to accommodate rapid on-demand PI availability at targeted locations of PI(4,5)P2 signaling and turnover in response to external cues, in dendrite development. Using state-of-the-art genetic probes to track PI(4,5)P2 and PIP3 signaling, combined with cutting edge genetic approaches including acute gene overexpression and knockdown in utero, CRISPR-mediated genome editing, and highly localized manipulations of signaling, this study is designed to determine the role of Nir2-activity in dendrite development in mouse developing cortical pyramidal neurons. The elaborate dendritic arbors of pyramidal neurons underlie synaptic connectivity. Disruption in dendrite development plays a causative role in many neurodevelopmental and psychiatric disorders, mental disability, epilepsy, Schizophrenia, Fragile X, and autism. Abnormal activity of Akt is associated with these pathologies and characterized by defects in dendrite development. Furthermore, Nir proteins are linked to human neuropathologies. By exploring the mechanistic basis of PI- signaling, this study will advance understanding of early molecular events in dendrite structural plasticity in principal neurons in the rodent brain and their implications in neurodevelopmental pathologies.
