Mechanisms of disease pathogenesis in Spinal Muscular Atrophy

Abstract

Low levels of survival motor neuron (SMN) protein cause the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA), through mechanisms that are poorly defined. SMN protein is ubiquitously expressed, however the major pathological hallmarks of SMA are focused on the neuromuscular system, including a loss of lower motor neurons in the ventral horn of the spinal cord and atrophy of skeletal muscle. At present there is no cure for SMA. Most research to date has focused on examining how low levels of SMN lead to pathological changes in motor neurons, therefore the contribution of other tissues, for example muscle, remains unclear. In this thesis I have used proteomic techniques to identify intrinsic molecular changes in muscle of SMA mice that contribute to neuromuscular pathology in SMA. I demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomics data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. In addition robust up-regulation of VDAC2 and down-regulation of parvalbumin was confirmed in two mouse models of SMA as well as in patient muscle biopsies. Thus intrinsic pathology of skeletal muscle is an important event in SMA. I then used proteomics to identify individual proteins in skeletal muscle of SMA that report directly on disease status. Two proteins, GRP75 and calreticulin, showed increased expression levels over time in different muscles as well as in skin samples, a more accessible tissue for biopsies in patients. Preliminary results suggest that GRP75 and calreticulin can be detected and measured in SMA patient muscle biopsies. These results show that proteomics provides a powerful platform for biomarker identification in SMA, revealing GRP75 and calreticulin as peripherally accessible potential protein biomarkers capable of reporting on disease progression in muscle as well as in skin samples. Finally I identified a role for ubiquitin-dependent pathways in regulating neuromuscular pathology in SMA. Levels of ubiquitin-like modifier activating enzyme 1 (UBA1) were reduced in spinal cord and skeletal muscle tissue of SMA mice. Dysregulation of UBA1 and subsequently the ubiquitination pathways led to the accumulation of β-catenin. I show here that pharmacological inhibition of β-catenin robustly ameliorates neuromuscular pathology in animal models of SMA. Interestingly, downstream disruption of β-catenin was restricted to the neuromuscular system in SMA mice. Pharmacological inhibition of β-catenin failed to prevent systemic pathology in organs. Thus disruption of ubiquitin homeostasis, with downstream consequences for β-catenin signalling, contributes to the pathogenesis of SMA, thereby highlighting novel therapeutic targets for this disease.