Signaling Through Girdin Underlies Excessive Cell Morphogenesis Resulting from Depletion of Neurodevelopmental Disorder-Related Neurexin-2

During development, neurexin-2 (NRXN2) is a cell adhesion molecule localized to presynaptic terminals as well as axonal shafts and immature neurites, where it participates in the regulation of neuronal cell morphogenesis. Given its critical role in early neuronal development, NRXN2 is considered a susceptibility gene product for neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD) and intellectual disability (ID). However, the intracellular signaling mechanisms linking NRXN2 deficiency to abnormal neuronal cell morphology remain unclear. Herein, we investigated the molecular basis of excessive cell morphogenesis induced by the knockdown of NRXN2 using the N1E-115 cell line, a model of neuronal morphogenesis characterized by neurite outgrowth. Silencing NRXN2 using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13 system resulted in a marked enhancement of process elongation. Mechanistically, we found that Girdin (also called GIV or CCDC88A), a non-receptor guanine nucleotide exchange factor for heterotrimeric G proteins, can mediate the excessive process length phenotype. Transfection of either the regulator of G protein signaling (RGS) domain of RGS3, a GTPase-activating protein for G proteins, or the G protein-binding domain of engulfment and cell motility 1 (ELMO1) rescued the excessive process formation. Similar results were obtained in primary cortical neurons. In addition, these interventions normalized downstream Rac1 activity in cells. Together, our findings elucidate Girdin signaling as a mediator of excessive neuronal process formation following NRXN2 knockdown, providing mechanistic insight into how the loss of function of NRXN2 leads to aberrant cell morphogenesis at least at the molecular and cellular levels. These results suggest that signaling through Girdin may contribute to the morphological abnormalities associated with NRXN2-related neurodevelopmental disorders.