Targeting Astrocytic Connexin 43 Mitigates Glutamate-Driven Motor Neuron Stress in Late-Onset Spinal Muscular Atrophy

5q-associated spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival of motor neuron-1 (SMN1) gene, leading to progressive muscle weakness, atrophy, and respiratory failure. Although traditionally considered a motor neuron (MN)–specific disease, recent research highlights the in-volvement of astrocytes, particularly in regulating extracellular glutamate levels and promoting motor neuron toxicity. Dysfunctional MN–astrocyte interactions and astrocytic glutamate dysregulation contribute to MN degeneration. We investigated the role of astrocytic gap junctions, focusing on connexin 43 (Cx43), a key protein in astrocytic communication. We employed a combination of in vivo and in vitro approaches, including a mouse model representative of late-onset SMA, human astrocytes derived from induced pluripotent stem cells, and murine astrocyte cultures. We assessed changes in expression and localization using genetic modification, immunostaining, Western blotting, and quantitative PCR. Functional outcomes were further investigated using ex vivo spinal cord slice cultures, Ca2+-imaging, and glutamate release assays. Our experiments revealed a significant upregulation of Cx43 in the spinal cord of late-onset SMA mice and in SMN-deficient murine and human induced astrocyte cultures. Elevated Cx43 expression was associated with increased astrocytic glutamate release and motor neuron (MN) tox-icity. Ca2+-imaging demonstrated that this enhanced glutamate release was mediated via Cx43-dependent mechanisms. Notably, pharmacological inhibition of Cx43 using Gap27, a Cx32 hemichannel blocker, in slice cultures attenuated glutamate release. It reduced MN Ca2+ response, supporting a causal role of Cx43 in mediating astrocyte-driven excitotoxicity in late-onset SMA. These findings suggest that astrocytic Cx43 contributes to gluta-mate-mediated MN toxicity in late-onset SMA. By identifying Cx43 as a contributor to astrocyte dysfunction, our work supports the growing recognition of non-neuronal con-tributions to late-onset SMA pathology.