Persistent deficits in the motor unit following mono and dual administration of SMN up-regulators in the SmnΔ7 mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is characterized by motor neuron loss and neuromuscular junction (NMJ) pathology. Although SMN-upregulating therapies such as Nusinersen markedly improve survival and motor function for many patients, impactful deficits often remain. In order to generate the next generation of therapy for SMA, it is critical that we understand the cellular basis for persistent deficits and find strategies to support and promote motor unit repair. Here we performed a detailed temporal analysis of the distal motor unit following administration of the Smn up-regulator Nusinersen in a range of differentially vulnerable cranial muscles in the SmnΔ7 mouse model. We show that early administration of Nusinersen facilitates progressive recovery of motor endplate innervation, even in the most vulnerable muscles. However, there is a persistent decrease in intramuscular motor axon number and increase in motor unit size, which is most severe in the most vulnerable muscles. We further show that combining Nusinersen with the Risdiplam tool compound SMN-C8 leads to a synergistic increase in Smn levels but does not produce broad improvements in motor unit recovery beyond those achieved with Nusinersen alone. Nevertheless, dual therapy resulted in significant improvement in hindlimb splay score from post-natal day 10 onwards. These effects suggest that enhanced SMN restoration may confer selective functional and structural benefits, although these were insufficient to fully rescue persistent motor unit pathology. Collectively, our findings demonstrate that early Smn restoration enables robust NMJ reinnervation but fails to prevent axon loss and motor unit remodelling. The limited additional benefit observed with dual SMN up-regulation, despite synergistic increases in Smn levels, suggests a potential ceiling effect for SMN-dependent rescue and highlights the need for adjunctive SMN-independent strategies aimed at preserving axons, stabilizing motor units, and promoting neuromuscular regeneration in SMA.