Abstract 1382 Neurons rely on local PIP2/ PIP3 homeostasis at ER-PM contact sites for proper dendrite development

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key phosphoinositide at the plasma membrane (PM) that mediates various downstream signaling which are essential during early neuronal development and specifically, dendrite maturation and arborization. These include the activation of phosphatidylinositol 3,4,5-triphosphate (PIP3)-dependent Akt signaling, which has been known to play a critical role in dendrite development. In the developing brain, neurons are perpetually exposed to diffusible extracellular cues, including neurotrophic growth factors that trigger the use of PI(4,5)P2, resulting in its rapid hydrolysis and depletion. Currently, little is known about how neurons regulate local PI(4,5)P2 homeostasis to enable sustained PI(4,5)P2/ PIP3 signaling at subcellular compartments, such as dendrite branching points and dendritic tips, during early developmental stages. Recent studies conducted primarily in cell-lines demonstrated that the lipid transport protein, Nir2, is a key regulator of PM PI(4,5)P2 replenishment and Akt signaling upon intense external cue stimulation by rapidly transporting specific lipid intermediates at ER-PM contact sites to accommodate the phosphatidylinositol (PI) cycle. We thus propose that local lipid transport mediated by Nir2 at ER-PM junctions is critical for maintaining PM PI(4,5)P2 homeostasis and PIP3-dependent Akt signaling, ultimately regulating dendrite growth and maturation during early neuronal development. Using short hairpin RNA (shRNA) to knockdown Nir2 expression in mouse cortical pyramidal neurons through in utero electroporation, we observe severe defects in dendrite development, elongation and branching. Combining the genetically encoded PIP3 biosensor, Akt-PH, with total internal reflection fluorescent (TIRF) microscopy, our data show that PM PIP3 availability upon stimulation with the neurotrophin brain-derived neurotrophic factor (BDNF) is reduced following Nir2 knockdown (KD) in cultured cortical neurons, implicating shrinkage in the PI(4,5)P2 pool. Furthermore, our biochemical examinations demonstrate that Nir2 KD significantly reduces the activities of Akt and GSK-3β, which are major effectors downstream of PIP3 production. These results suggest that Nir2 plays a critical role in dendrite development through the regulation of PI(4,5)P2/ PIP3-dependent signaling in developing neurons. We further observed robust Nir2 recruitment to the PM as distinct puncta upon PLC-mediated PI(4,5)P2 depletion following BDNF stimulation. Post-stimulation Nir2 puncta colocalize with the ER-PM junction marker, MAPPER, indicating that Nir2 responds to PM PI(4,5)P2 depletion and specifically translocates to ER-PM contact sites to facilitate local lipid exchange. Together, our study supports that Nir2 regulates dendrite development via local PI(4,5)P2 replenishment and subsequent PIP3-Akt signaling in developing neurons. This work was supported by grants from NIH, NS084111, NS114914, and NS119512