Drp1 in M1 layer V GABAergic neurons orchestrates rTMS-mediated motor restoration and analgesia after spinal cord injury.

Spinal cord injury (SCI) leads to chronic motor and sensory deficits, with progressive secondary neurodegeneration posing a major therapeutic challenge. Although high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) over the primary motor cortex (M1) shows therapeutic potential, its underlying cellular mechanisms remain poorly understood. This translational study first demonstrated that HF-rTMS concurrently improved motor function and alleviated neuropathic pain in retrospective clinical cohorts and a validated murine SCI model. To decipher the supraspinal mechanism, we used an integrated approach combining behavioral analyses, transmission electron microscopy and single-nucleus RNA sequencing. Transcriptomics revealed that HF-rTMS specifically rescued SCI-induced disruptions in oxidative phosphorylation and mitochondrial energy metabolism pathways within M1 GABAergic neurons. Here we pinpointed a key molecular lesion: SCI selectively downregulated the mitochondrial fission regulator Drp1 in M1 layer V GABAergic neurons, leading to dysfunctional mitochondrial dynamics and bioenergetic deficits. HF-rTMS restored Drp1 levels and mitochondrial ultrastructure specifically in M1 but not in the primary somatosensory cortex, underscoring its region-selective action. Most importantly, functional causality was established: Drp1 overexpression in M1 GABAergic neurons mimicked the therapeutic benefits of HF-rTMS, whereas Drp1 knockdown or its pharmacological inhibition completely abolished these effects. Our findings establish impaired mitochondrial dynamics in a specific cortical microcircuit as a convergent driver of multisystem deficits post SCI, and identify Drp1 as a pivotal molecular target of HF-rTMS. This work provides a novel mechanistic foundation for Drp1-directed precision therapies, highlighting the potential of rescuing cortical mitochondrial bioenergetics to halt progressive secondary damage and improve functional recovery after central nervous system injury. This study demonstrates that high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) alleviates motor dysfunction and neuropathic pain after spinal cord injury (SCI). We show that HF-rTMS acts by upregulating the mitochondrial fission protein Drp1 specifically in layer V GABAergic neurons of the primary motor cortex. This restoration of Drp1 rescues impaired mitochondrial dynamics, normalizes associated proteins (VDAC-1 and Tom20) and recovers cellular energy (ATP) production. Consequently, rTMS reverses SCI-induced mitochondrial pathology, leading to substantial functional recovery. The therapeutic effect is dependent on Drp1, as its inhibition abolishes the benefits of HF-rTMS. Our findings reveal a precise neuromodulation mechanism targeting cortical mitochondrial homeostasis for treating SCI comorbidity.