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

5q-associated Spinal Muscular Atrophy (SMA) is a hereditary neuromuscular disorder caused by mutations in the <i>survival of motor neuron 1</i> (<i>SMN1</i>) gene, leading to progressive muscle weakness, and atrophy. While traditionally viewed as a motor neuron (MN)-specific disease, emerging evidence highlights the critical role of astrocytes, particularly in regulating extracellular glutamate and mitigating MN toxicity. Here, we investigated astrocytic gap junctions with a focus on connexin 43 (Cx43). Using in vivo and in vitro approaches-including a late-onset SMA mouse model, human-derived astrocytes, and murine astrocyte cultures-we analyzed Cx43 expression and localization via genetic modification, immunostaining, Western blotting, and quantitative PCR. Functional consequences were assessed using ex vivo spinal cord slice cultures, Ca²⁺-imaging, and glutamate release assays. We found significant Cx43 upregulation in late-onset SMA mice, as well as in SMN-deficient murine and human-derived astrocytes. Increased Cx43 expression correlated with elevated astrocytic glutamate release and MN toxicity. Ca²⁺-imaging indicated Cx43-dependent mechanisms underlying this enhanced release. Pharmacological Cx43 inhibition with Gap27 reduced glutamate release and MN Ca²⁺ responses. These findings identify astrocytic Cx43 as a contributor to glutamate-mediated MN toxicity in late-onset SMA and support growing recognition of non-neuronal mechanisms in SMA pathology.