Spinal muscular atrophy (SMA) is an autosomal recessively inherited motor neuron disease characterized by progressive muscle weakness and atrophy(Lunn and Wang, 2008). Most cases of SMA are caused by homozygous mutation, deletion, or conversion of a telomeric copy of the survival motor neuron gene (SMN1 [MIM 600354]) (Lefebvre et al., 1995), which produces the SMN protein, essential for splicing of pre-mRNA and small nuclear ribonucleic particle biogenesis (Pellizzoni et al., 1998). The centromeric SMN gene (SMN2 [MIM 601627]) products are mainly unstable truncated SMN proteins lacking C-terminal residues attributed to alternative splicing. Therefore, the products of SMN2 are not sufficient to compensate for the SMN1 gene defect and to combat progressive motor neuron degeneration (Wirth, 2000). SMA model mice have been generated by mouse Smn knockout and human SMN2 transgenic methods (Hsieh-Li et al., 2000). These mice are useful for research on disease mechanisms and therapeutics.

Effective drugs for patients with SMA have been well developed and applied in clinical practice, including nusinersen (an antisense oligonucleotide [ASO]), risdiplam (an RNA targeting small molecule), and onasemnogene abeparvovec (a viral vector carrying the SMN1 gene) (Darras et al., 2021; Finkel et al., 2017; Mendell et al., 2017). All these drugs can robustly increase SMN expression in spinal motor neurons (Dominguez et al., 2011; Hua et al., 2011; Naryshkin et al., 2014), and subsequently eliminate disease progression and regain neurological functional development in children, especially initiated at the presymptomatic stage (Crawford et al., 2023; Finkel et al., 2023; Strauss et al., 2022). In SMA adults beyond the neurodevelopmental period, surprisingly, treatment with nusinersen can not only prevent neurological decline but improve motor functional behaviors (Cho et al., 2023; Coratti et al., 2021; Hagenacker et al., 2020). Regarding the mechanisms for such functional improvement, previous animal studies have demonstrated the therapeutic benefits of SMN-restoring ASO in SMA mice possibly via rescued motor unit maturation delays; however, the timing of treatment was either at the newborn stage (Arnold et al., 2016) or around the onset (Bogdanik et al., 2015) that the condition may not fully represent patients receiving treatment at adult or adolescent stage after decades of disease onset. Recent electrophysiological studies have shown the elevation of estimated motor unit numbers through increased sprouting and reinnervation at the neuromuscular junction after nusinersen treatment as the mechanisms behind such improvement (Kariyawasam et al., 2020; Schneider et al., 2021) but without pathological confirmation. Understanding the mechanisms of functional improvement after nusinersen treatment in SMA can provide knowledge for optimization of therapy and contribute insight for other neurodegenerative diseases. In this study, we analyzed a group of adult SMA patients to confirm functional and electrophysiological improvement after nusinersen treatment and then investigated the underlying therapeutic mechanisms behind such improvement using adult SMA mice at the late stage (12-month-old). Our results imply that some live but functionless motor neurons become functional, while some denervating neuromuscular junctions (NMJs) become innervating NMJs through reinnervation after nusinersen treatment.