Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by recessive pathogenic variants affecting the survival of motor neuron (SMN1) gene (localized on 5q). In consequence, cells lack expression of the corresponding protein. This pathophysiological condition is clinically associated with motor neuron (MN) degeneration leading to severe muscular atrophy. Additionally, vulnerability of other cellular populations and tissues including skeletal muscle has been demonstrated. Although the therapeutic options for SMA have considerably changed, treatment responses may differ thus underlining the persistent need for validated biomarkers. To address this need and to identify novel marker proteins for SMA, we performed unbiased proteomic profiling on cerebrospinal fluid derived (CSF) from genetically proven SMA type 1–3 cases and afterwards performed ELISA studies on CSF and serum samples to validate the potential of a novel biomarker candidates in both body fluids. To further decipher the pathophysiological impact of this biomarker, immunofluorescence studies were carried out on spinal cord and skeletal muscle derived from a 5q-SMA mouse model. Proteomics revealed increase of LARGE1 in CSF derived from adult patients showing a clinical response upon treatment with nusinersen. Moreover, LARGE1 levels were validated in CSF samples of further SMA patients (type 1–3) by ELISA. These studies also unveiled a distinguishment between groups in improvement of motor skills: adult patients do present with lowered level per se at baseline visit while no elevation upon treatment in the pediatric cohort can be observed. ELISA-based studies of serum samples showed no changes in the pediatric cohort but unraveled elevated level in adult patients responding to future intervention with nusinersen, while non-responders did not show a significant increase. Additional immunofluorescence studies of LARGE1 in MN and skeletal muscle of a SMA type 3 mouse model revealed an increase of LARGE1 during disease progression. Our combined data unraveled LARGE1 as a protein dysregulated in serum and CSF of SMA-patients (and in MN and skeletal muscle of SMA mice) holding the potential to serve as a disease marker for SMA and enabling to differentiate between patients responding and non-responding to therapy with nusinersen.

5q-related Spinal muscular atrophy (SMA) is a hereditary multi-systemic disorder leading to progressive muscle atrophy and weakness caused by the degeneration of spinal motor neurons (MNs) in the ventral horn of the spinal cord. Three SMN-enhancing drugs for SMA treatment are available. However, even if these drugs are highly effective when administrated early, several patients do not benefit sufficiently or remain non-responders, e.g., adults suffering from late-onset SMA and starting their therapy at advanced disease stages characterized by long-standing irreversible loss of MNs. Therefore, it is important to identify additional molecular targets to expand therapeutic strategies for SMA treatment and establish prognostic biomarkers related to the treatment response. Using high-throughput nCounter NanoString technology, we analyzed serum samples of late-onset SMA type 2 and type 3 patients before and six months under nusinersen treatment. Four genes (AMIGO1, CA2, CCL5, TLR2) were significantly altered in their transcript counts in the serum of patients, where differential expression patterns were dependent on SMA subtype and treatment response, assessed with outcome scales. No changes in gene expression were observed six months after nusinersen treatment, compared to healthy controls. These alterations in the transcription of four genes in SMA patients qualified those genes as potential SMN-independent therapeutic targets to complement current SMN-enhancing therapies.

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.

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.