A Noncanonical Neuroligin 3-Centered Complex Promotes Functional Recovery of Spinal Cord Injury

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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 complex for SCI repair.

1. Introduction

Spinal cord injury (SCI) typically results in permanent damage to sensorimotor function due to the very limited regeneration potential of neuronal networks in adult mammals. The functional recovery of SCI primarily relies on the appropriate reconstruction of synaptic communications between cortical projection and propriospinal neurons. A synapse is an intercellular complex consisting of apposed pre- and postsynaptic terminals, with a synaptic cleft between them, mediating the transmission of information between two neurons or between a neuron and its target cell. Synaptic cell adhesion molecules (CAMs) play pivotal roles in neuronal communication. They not only bridge adjacent pre- and postsynaptic specializations through homophilic or heterophilic interactions but also mediate trans-synaptic signaling across the synaptic cleft. Despite the extensive description of various trans-synaptic CAMs during development, the key players that facilitate the reestablishment of neuronal circuits after injury are not comprehensively understood.

Over the last few decades, a substantial body of evidence has highlighted the considerable potential of stem cell-based regenerative medicine in treating injuries and disorders of the central nervous system (CNS). Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are particularly promising for SCI treatment due to their easy accessibility, rapid self-renewal, low immunogenicity, and reduced tumorigenesis risk. The current understanding of MSCs' therapeutic effects underscores their immunomodulatory and paracrine activities, which contribute to neuroprotection, neurogenesis, and angiogenesis. While in vitro studies confirm MSCs' potential for neuronal transdifferentiation under specific conditions, its occurrence within the in vivo spinal cord microenvironment (SCE) and its contribution to functional recovery remain debated. This leads us to critical questions: Can the SCE induce neuronal reprogramming of transplanted MSCs? If so, do these activated neuron-associated genes possess neurorestorative functions? And can even expressing these key genes in injured neurons also promote neural circuit reconstruction? To answer these questions, we performed RNA-seq on hUC-MSCs pre- and post-transplantation in a rat model with or without sub-acute SCI. By doing so, we aimed to identify novel target genes that can promote the remodeling of damaged spinal cord neural connections.