Improving neuronal recovery in spinal cord injury with NEP1-40-modified neural stem cells through RhoA/ROCK signaling pathway modulation

To evaluate the therapeutic potential of NEP1-40-expressing neural stem cells (NSCs) in improving outcomes in spinal cord injury (SCI) by modulating the RhoA/ROCK signaling pathway. NEP1-40 was overexpressed by transfecting isolated NSCs with NEP1-40 lentivirus, and their differentiation following Nogo-A treatment was assessed via Tuj-1 immunofluorescence. The effects of NEP1-40 overexpression on axonal damage of differentiated NSCs and DRG neurons were examined using Tuj-1 and F-actin immunofluorescence, along with Western blotting for GAP-43, MAP-2, and APP. The activation of RhoA/ROCK and its downstream pathways, namely, PTEN/PI3K/AKT and MLC/LIMK, were analyzed by Western blotting. In a rat SCI model, following NSC transplantation, was assessed by examining neuronal regeneration markers. Furthermore, the Basso, Beattie, and Bresnahan (BBB) scale behavioral tests were used to assess motor function recovery. NEP1-40 overexpression inhibited the RhoA/ROCK signaling, followed by the stimulating the PI3K/AKT pathway and promoting neuronal differentiation under Nogo-A treatment. The inhibition of RhoA/ROCK signaling suppressed the MLC/LIMK pathway to enhance neurite outgrowth in NSC-differentiated and DRG neurons. In SCI rats, NEP1-40 overexpression increased the number of NSCs and neurons, whereas NEP1-40 overexpression in NSCs significantly increased the number of 5-HT-, ChAT-, and CGRP-positive neurons, as well as the levels of p-GAP-43, GAP-43 and MAP-2, and reduced the level of APP. These changes were accompanied by improved BBB scores. NEP1-40-modified NSCs enhance SCI recovery by activating the PI3K/AKT pathway and inhibiting the MLC/LIMK cascade, thereby promoting neuronal differentiation and reducing axonal damage. Altogether, this approach demonstrates promising therapeutic potential to improve NSC efficacy in SCI.