Sakers_2025_bioRxiv__

Astrocytes of the central nervous system have an intricate and highly branched morphology. Proper development of perisynaptic astrocyte processes is necessary for the tripartite synapse development, maturation, and function. However, how astrocyte morphogenesis is controlled, and the specific molecules orchestrating this development are largely unknown. Previously, we identified Neuroligins (NLs) 1-3 as regulators of astrocyte morphogenesis by forming transcellular adhesions with neuronal neurexins. Here, we found that NL2 plays a non-redundant essential role in astrocyte morphogenesis. Through structure and function studies, we identified a WW-binding motif within the NL2 intracellular domain required for astrocyte morphogenesis. Using cell-specific in vivo proximity labeling by biotinylation (iBioID), we found that each NL displays distinct protein-protein interactions within astrocytes, and NL2 has cell-type specific interactomes in astrocytes versus neurons. By leveraging this interactome, we conducted in vitro and in vivo candidate screens and identified WW domain-containing E3 ubiquitin ligase Nedd4l to control astrocyte morphogenesis. Biochemical assays revealed that Nedd4l ubiquitinates and stabilizes NL2, and this ubiquitination step is required for astrocyte morphogenesis. This study shows that Neuroligins have non-overlapping roles in controlling astrocyte growth and uncovers a molecular mechanism of how NL2 mediates astrocyte morphogenesis.