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.
Hideji Yako, Mikito Takahashi, Mami Akiyama et al.· International Journal of Mol...· 1 citation
Neuroligin-2 (NLGN2) is a cell adhesion molecule implicated in neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD) and intellectual disability (ID). While NLGN2 is well known as a postsynaptic organizer of predominantly inhibitory synapses, accumulating evidence suggests that NLGN family proteins are also involved in neuronal morphogenesis during early developmental stages. However, a gap remains in our understanding of how loss of function in NLGN2 potentially leads to abnormal neuronal morphogenesis. Here, we investigated the molecular basis of excessive neuronal process formation induced by depletion of NLGN2 using the N1E-115 cell line, an established model of neuronal differentiation. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13-mediated knockdown of NLGN2 promoted neuronal process elongation and neuronal differentiation marker expression. Mechanistically, NLGN2 knockdown resulted in activation of coiled-coil and hook domain-containing protein 88A (CCDC88A, also known as Girdin or GIV), a non-receptor guanine nucleotide exchange factor for heterotrimeric G proteins. Transfection of the regulator of G protein signaling (RGS) domain of RGS3, a negative regulator of G proteins, or the G protein-binding domain of engulfment and cell motility 1 (ELMO1) effectively decreased the excessive process elongation phenotype. Similar effects were observed in primary cortical neurons. Furthermore, these interventions normalized elevated Rac1 activity induced by NLGN2 knockdown. Collectively, our findings identify the CCDC88A-G protein-ELMO signaling pathway as a key mediator of excessive neuronal morphogenesis following NLGN2 knockdown. These results provide valuable insight into the mechanisms by which NLGN2 dysfunction may contribute to abnormal neuronal morphogenesis and suggest potential recovery strategies.
Hideji Yako, Mikito Takahashi, Shiori Tada et al.· International Journal of Mol...· 0 citations