5q-associated Spinal Muscular Atrophy (SMA) is a hereditary neuromuscular disorder caused by mutations in the survival of motor neuron 1 (SMN1) 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.

Background: The treatment landscape for spinal muscular atrophy (SMA) has changed significantly with the approval of gene-based therapies such as nusinersen for adults with SMA (pwSMA). Despite their efficacy, high costs and treatment burden highlight the need for biomarkers to objectify or predict treatment response. This study aimed to identify such biomarkers. Methods: A proteomic analysis of cerebrospinal fluid (CSF) from pwSMA (n = 7), who either significantly improved (SMA Improvers) or did not improve in motor function (SMA Non-Improvers) under nusinersen therapy, was performed. Data are available via ProteomeXchange with identifier PXD065345. Candidate biomarkers—Neuronal Pentraxin 2 (NPTX2), Contactin 5 (CNTN5), and Anthrax Toxin Receptor 1 (ANTXR1)—were investigated by ELISA in serum and CSF from an independent pwSMA cohort (n = 14) at baseline, 2 and 14 months after therapy initiation. Biomarker concentrations were correlated with clinical outcomes. Additionally, NPTX2 was stained in spinal cord sections from a mild SMA mouse model (FVB.Cg-Smn1tm1Hung Tg(SMN2)2Hung/J). Results: CSF NPTX2 levels decreased in pwSMA after 14 months of nusinersen therapy, independent of clinical response. The change in NPTX2 serum levels over 14 months of nusinersen treatment correlated with the change in HFMSE during this period. CNTN5 and ANTXR1 showed no significant changes. In the SMA mouse model, NPTX2 immunoreactivity increased at motoneuron loss onset. Conclusions: NPTX2 emerges as a potential biomarker of treatment response to nusinersen in pwSMA suggesting its significant pathophysiological role in late-onset SMA, warranting further investigation.

Spinal muscular atrophy (SMA) comprises a spectrum of clinical severities, yet the pathomechanisms of late-onset forms (Type III) remain insufficiently understood. While severe early-onset SMA has been extensively investigated using existing models, their translational relevance to adult disease is limited. Here, we recommend the 4-copy SMN2 mouse (FVB.Cg- Smn1 tm1Hung Tg( SMN2 )2Hung/J) as the most appropriate model for late-onset SMA. This model exhibits delayed onset, progressive motor dysfunction, and extended survival, enabling the study of chronic neurodegenerative processes, including astrocyte-mediated motor neuron pathology. Its prolonged therapeutic window makes the model suitable for mechanistic and translational investigations of late-onset SMA.

Background: The treatment landscape for spinal muscular atrophy (SMA) has changed significantly with the approval of gene-based therapies such as nusinersen for adults with SMA (pwSMA). Despite their efficacy, high costs and treatment burden highlight the need for biomarkers to objectify or predict treatment response. This study aimed to identify such biomarkers. Methods: A proteomic analysis of cerebrospinal fluid (CSF) from pwSMA (n = 7), who either significantly improved (SMA Improvers) or did not improve in motor function (SMA Non-Improvers) under nusinersen therapy, was performed. Data are available via ProteomeXchange with identifier PXD065345. Candidate biomarkers—Neuronal Pentraxin 2 (NPTX2), Contactin 5 (CNTN5), and Anthrax Toxin Receptor 1 (ANTXR1)—were investigated by ELISA in serum and CSF from an independent pwSMA cohort (n = 14) at baseline, 2 and 14 months after therapy initiation. Biomarker concentrations were correlated with clinical outcomes. Additionally, NPTX2 was stained in spinal cord sections from a mild SMA mouse model (FVB.Cg-Smn1tm1Hung Tg(SMN2)2Hung/J). Results: CSF NPTX2 levels decreased in pwSMA after 14 months of nusinersen therapy, independent of clinical response. In serum, NPTX2 levels were lower in SMA Improvers compared to Non-Improvers at 14 months. CNTN5 and ANTXR1 showed no significant changes. In the SMA mouse model, NPTX2 immunoreactivity increased at motoneuron loss onset. Conclusion: NPTX2 emerges as a potential biomarker of treatment response to nusinersen in pwSMA suggesting its significant pathophysiological role in late-onset SMA, warranting further investigation.

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.

Spinal muscular atrophy (SMA) is increasingly recognized as a multisystem disorder involving non-neuronal cells, yet the role of Schwann cells (SCs) in late-onset SMA (loSMA) remains unclear. We investigated age-dependent peripheral nerve pathology in a four-copy SMN2 mouse model of loSMA. Sciatic nerves from wild-type and loSMA mice were analyzed at postnatal (P) days 20, 35, 70, and >100 using semi-thin morphometry, immunofluorescence for MBP, Sox10, Sox2, and F4/80, and nerve conduction studies. loSMA nerves showed reduced myelin thickness at all time points and smaller axon diameters at P20 and P35. G- ratios were reduced at P20 but increased from P35 onward, indicating progressively altered axon-myelin relationships. MBP immunofluorescence intensity, compound muscle action potential amplitude, and nerve conduction velocity were reduced in loSMA mice at P>100. The proportion of Sox2 ⁺ SCs increased from P35 onward, while Sox10 ⁺ cell abundance increased at later stages. F4/80 ⁺ macrophages were transiently elevated at P35 and correlated with Sox2 ⁺ cell numbers at this stage. These findings demonstrate age-dependent myelin abnormalities, altered SC states, and transient accumulation of macrophages in loSMA peripheral nerves. Whether these changes are SC-autonomous or secondary to chronic axonal dysfunction remains to be determined.