HUC-MSC-mediated Recovery Of Subacute Spinal Cord Injury Through Enhancing The Pivotal Subunits β3 And γ2 Of The GABA_A Receptor

Background and ObjectiveSpinal cord injurie(SCI)cause permanent and severe nerve damage leading to paralysis of limbs,sensory loss,and incontinence.However,the current clinical treatment methods are limited,and SCI remains an incurable neurological disease.In recent years,cell transplantation has shown good results in the treatment of SCI,among which human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)are particularly attractive,which play a role in immunomodulation,anti-inflammatory,anti-apoptotic,and axonal promotion.It has excellent effects in aspects such as remyelination and remyelination.However,the detailed mechanisms underlying hUC-MSC-mediated functional recovery of SCI have not been fully elucidated.Therefore,it is imperative to explore the potential targets of hUC-MSCs for treatment SCI and to provide potencial drug targets for clinical treatment.Methods and ResultsAdult female rats suffering from subacute incomplete thoracic SCI were treated with intrathecal transplantation of hUC-MSCs.The beneficial effects of hUC-MSCs on SCI repair were evaluated by a series of behavioral analyses,motor evoked potentials(MEPs)recording of hindlimb and immunohistochemistry.We carried out extensive transcriptome comparative analyses of spinal cord tissues at the lesion site from the subacute phase of SCI(sub-SCI)either treated without(+PBS)or with hUC-MSCs(+MSC)at 0(sub-SCI),1,2,and 4 weeks post-transplantation(wpt),as well as normal spinal cord segments of intact/sham rats(Intact).Comparative transcriptome analysis revealed the GABAergic synapse pathway is significantly enriched as a main target of hUC-MSC-activated genes in the injured spinal cord.Adeno-associated virus(AAV)-mediated neuron-specific expression system was employed to functionally screen specific y-aminobutyric acid type A receptor(GABAAR)subunits promoting the functional recovery of SCI in vivo.Functional screening of the primary GABAAR subunits uncovered that Gabrb3 and Garbg2 harbored the motor and electrophysiological recovery-promoting competence.Moreover,targeting either of the two pivotal subunits β3 or γ2 in combination with/without the K+/Cl-cotransporter 2(KCC2)reinforced the therapeutic effects.The mature cortical axon scrape assay and transplantation of genetically modified MSCs with either overexpression or knockdown of brain-derived neurotrophic factor(BDNF)were employed to demonstrate that hUC-MSCs ameliorated the reduction of GABAAR subunits in the injured spinal cord via BDNF secretion in vitro and in vivo,respectively.Mechanistically,BDNF secreted by hUC-MSCs contributed to the upregulation of GABAAR subunits(β3&γ2)and KCC2 in the injured neurons.ConclusionsOur study identifies a novel mode for hUC-MSC-mediated locomotor recovery of SCI through synergistic upregulation of GABAAR β3 and γ2 along with KCC2 by BDNF secretion,indicating the significance of restoring the excitation/inhibition balance in the injured neurons for the reestablishment of neuronal circuits.This study also provides a potential combinatorial approach by targeting the pivotal subunit β3 or y2 and KCC2,opening up possibilities for efficacious drug design.