Phosphatidylinositol (3,5)-bisphosphate machinery regulates neurite thickness through neuron-specific endosomal protein NSG1/NEEP21

Phosphatidylinositol (3,5)-bisphosphate [PtdIns(3,5)P(2)] is a critical signaling phospholipid involved in endolysosome homeostasis. It is synthesized by a protein complex composed of PIKfyve, Vac14, and Fig4. Defects in PtdIns(3,5)P(2) synthesis underlie a number of human neurological disorders, including Charcot-Marie-Tooth disease, child onset progressive dystonia, and others. However, neuron-specific functions of PtdIns(3,5)P(2) remain less understood. Here, we show that PtdIns(3,5)P(2) pathway is required to maintain neurite thickness. Suppression of PIKfyve activities using either pharmacological inhibitors or RNA silencing resulted in decreased neurite thickness. We further find that the regulation of neurite thickness by PtdIns(3,5)P(2) is mediated by NSG1/NEEP21, a neuron-specific endosomal protein. Knockdown of NSG1 expression also led to thinner neurites. mCherry-tagged NSG1 colocalized and interacted with proteins in the PtdIns(3,5)P(2) machinery. Perturbation of PtdIns(3,5)P(2) dynamics by overexpressing Fig4 or a PtdIns(3,5)P(2)-binding domain resulted in mislocalization of NSG1 to nonendosomal locations, and suppressing PtdIns(3,5)P(2) synthesis resulted in an accumulation of NSG1 in EEA1-positive early endosomes. Importantly, overexpression of NSG1 rescued neurite thinning in PtdIns(3,5)P(2)-deficient CAD neurons and primary cortical neurons. Our study uncovered the role of PtdIns(3,5)P(2) in the morphogenesis of neurons, which revealed a novel aspect of the pathogenesis of PtdIns(3,5)P(2)-related neuropathies. We also identified NSG1 as an important downstream protein of PtdIns(3,5)P(2), which may provide a novel therapeutic target in neurological diseases.