The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. Here, we determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, we observed widespread perturbations in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (UBA1). SMN physically interacted with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMAlike neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways led to β-catenin accumulation, and pharmacological inhibition of β-catenin robustly ameliorated neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of β-catenin was restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of β-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. Our data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent β-catenin signaling, highlighting ubiquitin homeostasis and β-catenin as potential therapeutic targets for SMA.

The autosomal recessive neuromuscular disease spinal muscular atrophy (SMA) is caused by loss of survival motor neuron (SMN) protein. Molecular pathways that are disrupted downstream of SMN therefore represent potentially attractive therapeutic targets for SMA. Here, we demonstrate that therapeutic targeting of ubiquitin pathways disrupted as a consequence of SMN depletion, by increasing levels of one key ubiquitination enzyme (ubiquitin-like modifier activating enzyme 1 [UBA1]), represents a viable approach for treating SMA. Loss of UBA1 was a conserved response across mouse and zebrafish models of SMA as well as in patient induced pluripotent stem cell–derive motor neurons. Restoration of UBA1 was sufficient to rescue motor axon pathology and restore motor performance in SMA zebrafish. Adeno-associated virus serotype 9–UBA1 (AAV9-UBA1) gene therapy delivered systemic increases in UBA1 protein levels that were well tolerated over a prolonged period in healthy control mice. Systemic restoration of UBA1 in SMA mice ameliorated weight loss, increased survival and motor performance, and improved neuromuscular and organ pathology. AAV9-UBA1 therapy was also sufficient to reverse the widespread molecular perturbations in ubiquitin homeostasis that occur during SMA. We conclude that UBA1 represents a safe and effective therapeutic target for the treatment of both neuromuscular and systemic aspects of SMA.

Low levels of survival of motor neuron (SMN) protein lead to spinal muscular atrophy (SMA). The major pathological hallmark of SMA is a loss of lower motor neurons from spinal cord and peripheral nerve. However, recent studies have revealed pathological changes in other cells and tissues of the neuromuscular system. Here, we demonstrate intrinsic, SMN-dependent defects in Schwann cells in SMA. Myelination in intercostal nerves was perturbed at early- and late-symptomatic stages of disease in two mouse models of SMA. Similarly, maturation of axo-glial interactions at paranodes was disrupted in SMA mice. In contrast, myelination of motor axons in the corticospinal tract of the spinal cord occurred normally. Schwann cells isolated from SMA mice had significantly reduced levels of SMN and failed to express key myelin proteins following differentiation, likely due to perturbations in protein translation and/or stability rather than transcriptional defects. Myelin protein expression was restored in SMA Schwann cells following transfection with an SMN construct. Co-cultures of healthy neurons with diseased Schwann cells revealed deficient myelination, suggestive of intrinsic defects in Schwann cells, as well as reduced neurite stability. Alongside myelination defects, SMA Schwann cells failed to express normal levels of key extracellular matrix proteins, including laminin α2. We conclude that Schwann cells require high levels of SMN protein for their normal development and function in vivo, with reduced levels of SMN resulting in myelination defects, delayed maturation of axo-glial interactions and abnormal composition of extracellular matrix in peripheral nerve.

After the placebo-controlled extension of the pivotal US trial of glatiramer acetate for the treatment of relapsing multiple sclerosis ended, 208 participants entered an open-label, long-term treatment protocol Magnetic resonance imaging (MRI) was added to the planned evaluations of these subjects to determine the consequences of long-term treatment on MRI-defined pathology and evaluate its clinical correlates. Of the 147 subjects that remained on long-term follow-up, adequate images were obtained on 135 for quantitative MRI analysis. The initial imaging sessions were performed between June 1998 and January 1999 at 2,447 +/- 61 days (mean +/- standard deviation) after the subject's original randomization. Clinical data from a preplanned clinical visit were matched to MRI within 3 +/- 51 days. At imaging, 66 patients originally randomized to placebo (oPBO) in the pivotal trial had received glatiramer acetate for 1,476 +/- 63 days, and 69 randomized to active treatment with glatiramer acetate (oGA) were on drug for 2,433 +/- 59 days. The number of documented relapses in the 2 years prior to entering the open-label extension was higher in the group originally randomized to placebo (oPBO=1.86 +/- 1.78, oGA=1.03 +/- 1.28; P=0.002). The annualized relapse rate observed during the open-label study was similar for both groups (oPBO=0.2 7, +/- 0.45 oGA=0.28 +/- 0.40), but the reduction in rate from the placebo-controlled phase was greater for those beginning therapy with GA (oPBO reduced by 0.66 +/- 0.71, oGA reduced by 0.23 +/- 0.58; P=0.0002). One or more gadolinium enhancing lesions were found in 27.4% of all patients (number of distinct enhancements=1.16 +/- 2.52, total enhanced tissue volume=97 +/- 26 microl). The risk of having an enhancement was higher in those with relapses during the open-label extension (odds ratio 4.65, 95% confidence interval (CI) 2.0 to 10.7; P=0.001). The odds for finding an enhancement was 2.5 times higher for those patients originally randomized to placebo (CI 1.1 to 5.4; P=0.02) compared to those always on glatiramer acetate. MRI-metrics indicative of chronic pathology, particularly measures of global cerebral tissue loss (atrophy), were uniformly worse for those originally on placebo. These observations enrich our long-term follow up of the clinical consequences of treatment with glatiramer acetate to include its apparent effects on MRI-defined pathology. They show that the effect of glatiramer acetate on enhancements is definite, but modest, consistent with the drug's described mechanisms of action, and that a delay in initiating treatment results in progression of MRI-measured pathology that can be prevented.

BackgroundSpinal muscular atrophy (SMA) is a neuromuscular disease resulting from mutations in the survival motor neuron 1 (SMN1) gene. Recent breakthroughs in preclinical research have highlighted several potential novel therapies for SMA, increasing the need for robust and sensitive clinical trial platforms for evaluating their effectiveness in human patient cohorts. Given that most clinical trials for SMA are likely to involve young children, there is a need for validated molecular biomarkers to assist with monitoring disease progression and establishing the effectiveness of therapies being tested. Proteomics technologies have recently been highlighted as a potentially powerful tool for such biomarker discovery.MethodsWe utilized label-free proteomics to identify individual proteins in pathologically-affected skeletal muscle from SMA mice that report directly on disease status. Quantitative fluorescent western blotting was then used to assess whether protein biomarkers were robustly changed in muscle, skin and blood from another mouse model of SMA, as well as in a small cohort of human SMA patient muscle biopsies.ResultsBy comparing the protein composition of skeletal muscle in SMA mice at a pre-symptomatic time-point with the muscle proteome at a late-symptomatic time-point we identified increased expression of both Calreticulin and GRP75/Mortalin as robust indicators of disease progression in SMA mice. We report that these protein biomarkers were consistently modified in different mouse models of SMA, as well as across multiple skeletal muscles, and were also measurable in skin biopsies. Furthermore, Calreticulin and GRP75/Mortalin were measurable in muscle biopsy samples from human SMA patients.ConclusionsWe conclude that label-free proteomics technology provides a powerful platform for biomarker identification in SMA, revealing Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers capable of reporting on disease progression in samples of muscle and skin.