| Summary: | <p>Spinal muscular atrophy (SMA) a neuromuscular disorder caused by loss of the survival motor neuron (SMN) protein and characterized by motor neuron loss and muscle atrophy. Recent clinical trials of SMN restoration therapies changed the SMA therapeutic landscape and led to the first approval of a treatment. SMN restoration clearly has a positive impact on SMA pathology but is not a cure; patients will now live longer with a less severe form of SMA, leading to a growth of the patient population with additional therapeutic needs. Novel second-generation therapies are therefore to alleviate SMA related muscle pathology that can be used in combination with SMN restoration treatments. In the first part of the thesis, an unbiased approach was applied in which proteomic and transcriptomic analyses of Smn-depleted and SMN-restored SMA skeletal muscle were used to investigate dysregulated pathways, presenting the opportunity to identify novel muscle-specific treatments. Proteomic and transcriptomic analyses performed on skeletal muscle from pre-symptomatic and symptomatic Smn<sup>-/-</sup>;SMN2<sup>+/-</sup> and WT mice firstly show that SMN restoration completely normalizes protein and transcript expression in Smn<sup>-/-</sup>;SMN2<sup>+/-</sup> mice. Next, Connectivity Map (CMap), a drug repositioning strategy using publicly available databases, was employed to analyze proteomic and transcriptomic data and identify treatments able to restore aberrant pathways in SMA muscle. CMap analyses on ‘omics studies provided an extensive list of potential treatments for SMA, including some already tested in clinical and preclinical studies and new options, such as harmine. Reference genes produced by the CMap analysis for harmine shows certain genes to be upregulated and others downregulated in SMA mouse tissues and harmine treatment can reverse these expressions in various cell lines and Smn<sup>-/-</sup>;SMN2<sup>+/-</sup> mice. Importantly, harmine treatment significantly increases lifespan in severe Smn<sup>-/-</sup>;SMN2<sup>+/-</sup> and moderate Smn<sup>2B/-</sup> mice, rescues myofiber size and prevents motor neuron death. In addition to validating CMap as a method for identifying new treatments for SMA, while using harmine as a proof-of-concept we also generated an extensive list of drugs that can similarly be evaluated. Next, a biased approach was applied in which the TWEAK/Fn14 pathway, critical to the regulation of denervation-induced muscle atrophy and muscle proliferation, differentiation and metabolism, was investigated in SMA, since SMA is typified by neurodegeneration, muscle atrophy and metabolic perturbations. We investigated the expression profile of Tweak and Fn14 together with downstream metabolic effectors in SMA muscle and found significantly decreased levels during disease progression in Smn-/-;SMN2<sup>+/-</sup> and Smn<sup>2B/-</sup> mice. Using C2C12 myoblasts and human myoblasts, we confirmed a regulated expression of the TWEAK/Fn14 pathway during differentiation, which is dysregulated in SMA fibroblasts. siRNA mediated knockdown in C2C12s suggests a role for SMN in the aberrant expression of the TWEAK/Fn14 pathway in SMA. Overlap in dysregulation of myopathy, myogenesis and glucose metabolism pathways in SMA mice and TWEAK and Fn14 KO mice reinforced this finding. Through pharmacological interventions that target TWEAK/Fn14 pathway signaling, SMA disease phenotypes were improved in two distinct SMA mouse models. Taken together, these findings show that both biased and unbiased approaches can be used in order to identify modulators of muscle pathology in SMA as well as demonstrates that new investigative approaches, such as CMap, can augment the progress made by SMN restoration therapies.</p>
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