Neuroligin 3 promotes functional recovery of spinal cord injury through coordinating with Sar1a and Hspa8 at synapses

The recovery of locomotor function following incomplete spinal cord injury (SCI) primarily relies on the precise reconstruction of synaptic communications between spared corticospinal tract (CST) axons and propriospinal neurons. However, components capable of rebuilding synaptic machinery after injury remain elusive. Here, using RNA-Seq of human umbilical cord-derived MSCs (hUC-MSCs) before and after transplantation in SCI rats, along with in vitro and in vivo functional screenings, we identified Neuroligin 3 (NLGN3) as a spinal cord microenvironment (SCE)-activated cell adhesion molecule (CAM) in hUC-MSCs, promoting MSC-mediated functional recovery of SCI. Importantly, spinal neuron-specific activation of Nlgn3 led to significant functional improvement in SCI rats, resembling the effects of hUC-MSC transplantation. We elucidated the protein interactome of Nlgn3, enriched with proteins involved in synaptic transmission. Specifically, Nlgn3 interacted with synaptic vesicle (SV)-associated proteins, Sar1a and Hspa8, modulating their recruitment at synapses. Remarkably, restoring either of these two proteins in injured spinal neurons improved locomotor recovery of SCI, with further enhancement when combined with Nlgn3. Thus, our results not only reveal the previously unknown therapeutic effect of Nlgn3 in SCI repair but also propose a novel approach for treating SCI by targeting protein complexes centered around Nlgn3. This study also suggests that MSCs can serve as a stem cell platform for identifying innovative therapeutic targets and mechanisms to reestablish corticospinal-spinal relay circuits after SCI. In this study, 10-mm spinal cord segments centered at the epicenter were collected from rats subjected to different treatments for total RNA extraction. A total of 20 sequencing libraries were prepared, encompassing three biological replicates for MSC-in vitro, four replicates for the MSC-SCI-1d, MSC-sham-1d and MSC-sham-3d groups, and five replicates for the MSC-SCI-3d groups

不完全性脊髓损伤（incomplete spinal cord injury, SCI）后运动功能的恢复，主要依赖于保留的皮质脊髓束（corticospinal tract, CST）轴突与脊髓固有神经元之间突触通讯的精准重建。然而，损伤后可用于重建突触结构的相关成分仍尚未明确。
本研究通过对移植入脊髓损伤大鼠前后的人脐带源间充质干细胞（human umbilical cord-derived MSCs, hUC-MSCs）进行RNA测序（RNA-Seq），结合体外与体内功能筛选实验，鉴定出神经连接蛋白3（Neuroligin 3, NLGN3）是hUC-MSCs中被脊髓微环境（spinal cord microenvironment, SCE）激活的细胞黏附分子（cell adhesion molecule, CAM），可促进间充质干细胞介导的脊髓损伤功能恢复。
值得注意的是，脊髓神经元特异性激活Nlgn3可使脊髓损伤大鼠获得显著的功能改善，其效果与hUC-MSCs移植相仿。
本研究解析了Nlgn3的蛋白质相互作用组，该组富集了大量参与突触传递的蛋白质。具体而言，Nlgn3可与突触囊泡（synaptic vesicle, SV）相关蛋白Sar1a及Hspa8相互结合，调控二者在突触部位的招募。
尤为关键的是，在损伤的脊髓神经元中恢复这两种蛋白中的任意一种，即可改善脊髓损伤大鼠的运动功能恢复，若与Nlgn3联合应用则可进一步强化治疗效果。
因此，本研究结果不仅揭示了此前未被认知的Nlgn3在脊髓损伤修复中的治疗作用，还提出了一种以Nlgn3为核心靶向蛋白质复合物的脊髓损伤全新治疗策略。
此外，本研究还证实间充质干细胞可作为干细胞研究平台，用于筛选脊髓损伤后重建皮质脊髓-脊髓中继环路的创新治疗靶点与相关机制。
本研究中，我们从接受不同处理的大鼠体内采集了以损伤中心为中心的10mm脊髓节段，用于总RNA提取。最终共构建20个测序文库，其中：MSC-in vitro组设置3个生物学重复，MSC-SCI-1d组、MSC-sham-1d组及MSC-sham-3d组各设置4个生物学重复，MSC-SCI-3d组设置5个生物学重复。