A noncanonical neuroligin 3-centered complex promotes functional recovery of spinal cord injury

While human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold great potential for spinal cord injury (SCI) treatment, their intrinsic mechanisms are not fully understood. Given their multipotency, especially their neuronal transdifferentiation potential, we hypothesized that investigating the in situ transcriptional reprogramming of transplanted hUC-MSCs within the spinal cord microenvironment (SCE) could shed light on crucial genes for SCI repair. DiD-labeled hUC-MSCs were intrathecally transplanted in rats with or without sub-acute spinal cord bilateral hemisection injury and subsequently retrieved for RNA-seq. Comparative analysis of the transcriptomes of hUC-MSCs and functional screenings in vitro and in vivo, including heterologous synapse formation assay, transplantation of MSCs with gene overexpression or knockdown, AAV-mediated neuron-specific gene expression in SCI rats, behavioral tests, and motor evoked potentials (MEPs) were performed to identify the novel target gene Neuroligin 3 (Nlgn3). Immunoprecipitation followed by mass spectrometry (IP-Mass spec), cell aggregation assay, and immuno-electron microscopy were used to reveal the functional interacting partners of Nlgn3. Moreover, RT-qPCR, western blotting, immunofluorescence staining, and co-IP were used to elucidate the underlying mechanism. Using RNA-seq and functional screening, we identified NLGN3 as a neuronal cell adhesion molecule (CAM) activated by the SCE in transplanted hUC-MSCs to promote therapeutic efficacy. Critically, the neuron-specific restoration of Nlgn3 in the injured spinal cord alone was sufficient to achieve a comparable therapeutic effect. Mechanistically, Nlgn3 recruits the synaptic vesicle proteins Sar1a and Hspa8 to modulate synaptic strength. The combinatorial restoration of Nlgn3 with either Sar1a or Hspa8 synergistically enhanced SCI repair, highlighting the functional importance of this noncanonical Nlgn3-Sar1a-Hspa8 axis. This work unveils a novel therapeutic role for Nlgn3 in SCI treatment, demonstrating its ability to both enhance MSC transplantation efficacy and directly promote neural circuit reconstruction. We also propose a combinatorial strategy of targeting the noncanonical Nlgn3-centered protein complex. Furthermore, our study provides a valuable framework for uncovering novel therapeutic targets by investigating the transcriptional reprogramming of transplanted MSCs within injury microenvironments.