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Spinal Muscular Atrophy (SMA) is a childhood form of motor neuron disease that causes a progressive paralysis that, in its most severe form, results in death before two years of age. There is currently no cure or treatment for SMA. SMA is caused by a reduction in levels of Survival Motor Neuron (SMN) protein, which results in the degeneration of lower motor neurons. This degeneration is first observed at the neuromuscular junction (NMJ), where pre-synaptic nerve terminals belonging to the motor neuron become dysfunctional and degenerate during the early stages of disease. Several previous studies have shown that the some populations of motor neurons appear to have a resistance to SMA pathology, while other neighbouring populations are vulnerable. In this study, we attempted to elucidate the cause of this vulnerability spectrum. Initially, we characterised the relative vulnerability of ten different motor unit pools in an established mouse model of severe SMA and attempted to correlate these vulnerabilities with quantified aspects of motor unit morphology. From this study, no significant correlation could be found with any aspect of motor unit morphology examined, suggesting that morphological parameters of motor neurons do no influence their relative susceptibility. We then attempted to identify changes in basal gene expression between protected and vulnerable pools of motor units using microarray analysis. Motor unit pools were labelled using a retrograde tracer injected into muscles that had previously been identified as having highly vulnerable or resistant motor units. Labelled motor neuron cell bodies were then isolated from the spinal cord using laser capture micro-dissection and RNA was extracted for microarray analysis. From this study, we identified several molecular pathways and individual genes whose expression levels compared the gene expression profiles of vulnerable and resistant motor units. Thus, molecular differences between motor neuron pools likely underlie their relative vulnerability to degeneration in SMA. Lastly, we attempted to identify a novel peptide that could be used to label synapses, including neuromuscular junctions, in vivo. This would allow us to non-invasively visualise degenerating NMJs and other synapses in human patients without the need for a biopsy. Such a tool would be extremely valuable in assessing the effectiveness of drug trials, both in human patients and animal models, and may also contribute to earlier diagnosis of motor neuron disorders. To identify a potentially suitable peptide, we used a phage display library and panned for peptides that specifically bound to the outer surface of synapses using synaptosome preparations. From this panning we successfully enriched two peptides, the sequences of which were used to manufacture fluorescently tagged peptides.

Mounting evidence suggests that synaptic connections are early pathological targets in many neurodegenerative diseases, including motor neuron disease. A better understanding of synaptic pathology is therefore likely to be critical in order to develop effective therapeutic strategies. Spinal muscular atrophy (SMA) is a common autosomal recessive childhood form of motor neuron disease. Previous studies have highlighted nerve- and muscle-specific events in SMA, including atrophy of muscle fibres and postsynaptic motor endplates, loss of lower motor neuron cell bodies and denervation of neuromuscular junctions caused by loss of pre-synaptic inputs. Here I have undertaken a detailed morphological investigation of neuromuscular synaptic pathology in the Smn-/- ;SMN2 and Smn-/-;SMN2;Δ7 mouse models of SMA. Results imply that synaptic degeneration is an early and significant event in SMA, with progressive denervation and neurofilament accumulation being present at early symptomatic time points. I have identified selectively vulnerable motor units, which appear to conform to a distinct developmental subtype compared to more stable motor units. I have also identified significant postsynaptic atrophy which does no correlate with pre-synaptic denervation, suggesting that there is a requirement for Smn in both muscle and nerve and pathological events can occur in both tissues independently. Rigorous investigation of lower motor neuron development, connectivity and gene expression at pre-symptomatic time points revealed developmental abnormalities do not underlie neuromuscular vulnerability in SMA. Equivalent gene expression analysis at end-stage time points has implicated growth factor signalling and extracellular matrix integrity in SMA pathology. Using an alternative model of early onset neurodegeneration, I provide evidence that the processes regulating morphologically distinct types of synaptic degeneration are also mechanistically distinct. In summary, in this work I highlight the importance and incidence of synaptic pathology in mouse models of spinal muscular atrophy and provide mechanistic insight into the processes regulating neurodegeneration.

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.

The effects of denervation, non-weight bearing (unloading) or immobilization on hindlimb muscle growth, protein and amino acid metabolism were studied. In the first 3 days after denervation or unloading, atrophy of the soleus was caused by a suppression of protein synthesis and an acceleration of protein degradation. Thereafter, further atrophy, up to 6 days was due to depressed protein synthesis only. The changes in both protein synthesis and degradation in the first three days accounted for 69% and 65%, respectively, of the total loss of protein and mass in 6 days of unloaded or denervated soleus. Over the 6-day period, denervated soleus lost more mass and protein than the unloaded muscle owing to the earlier onset and greater extent of proteolysis. In denervated soleus, both lysosomal and non-lysosomal proteolysis may be enhanced, whereas in the unloaded muscle possibly only non-lysosomal proteolysis was enhanced. In both cases non-lysosomal proteolysis may be mediated by Ca²⁺-activated neutral protease, partially as a result of Ca²⁺ release from sarcoplasmic reticulum. Possibly due to the lack of lysosomal proteolysis, the insulin receptor did not show apparent increased turnover with unloading, as suggested by increased insulin sensitivity of in vitro protein turnover in the unloaded soleus. In contrast, denervated soleus showed a normal response to insulin for in vitro protein turnover. These findings suggested a mechanistic difference of unloading and denervation atrophy of soleus. A decreased ratio of glutamine/glutamate in fresh muscle suggested that the synthesis of glutamine in soleus may be diminished by denervation just as by unloading. This diminution of glutamine synthesis was probably due to reduced availability of ammonia, as evidenced by the slow disappearance of ATP in incubated denervated soleus. Similiar to unloading, denervation led to a decrease in aspartate concentration. This decreased concentration apparently resulted in decreased rather than increased utilization of aspartate. Effects of stretch on unloaded soleus were particularly pronounced in the first two days. Thereafter, in the stretched, unloaded soleus protein degradation increased to nearly the same extent as did protein synthesis. Hence after two days, stretch seems to lose its effectiveness in mitigating the effects of unloading so that it may not be an adequate preventive measure of muscle wasting under non-weight bearing condition.

No description available.

Rotator cuff injury is a debilitating condition and when injury cannot be managed through non-operative procedures, surgery is required. To determine explanations for the failure of surgery to restore joint function, human muscle biopsies of supraspinatus were studied compared to deltoid. Histology showed atrophy and a tendency toward fibrosis/fatty infiltration in injured supraspinatus. Findings from AChR-subunit western blot and Sema3A localization around satellite cells suggest supraspinatus denervation. Nucleotide incorporation to quantify satellite cell activation in culture showed a significant increase in BrdU+(active) satellite cells in supraspinatus treated with a nitric oxide-donor drug, but not deltoid muscle. The application of principal component analysis to these data extracted components that suggest variables assaying muscle atrophy, satellite cell activity, and fibrosis contribute strongly to the observed variability. The results suggest supraspinatus muscle of the injured rotator cuff is atrophic, denervated, possibly subject to fibro-fatty infiltration, and support the idea that treatment could promote growth in atrophic supraspinatus to improve functional outcomes. / October 2015

Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disorder characterised by lower motor neuron degeneration and paralysis. Although it is a leading genetic cause of childhood death no approved treatment options currently exist. As SMA is caused by low levels of the survival motor neuron (SMN) protein the majority of therapeutic strategies under development are therefore aimed at trying to elevate SMN levels. However, a number of limitations with these approaches exist demonstrating a need for the investigation of SMN-independent therapeutics. Of these non-classical pathways, the ubiquitin proteasome system (UPS) is an exciting new area of SMA research. The UPS is a system which degrades unwanted or damaged proteins and alterations in the UPS (including reduced levels of ubiquitin-like modifier activating enzyme 1 [Uba1] and increased levels of ubiquitin carboxyl-terminal esterase L1 [Uchl1] and β-catenin) have been recently identified in the neuromuscular system of SMA mice, providing promising new targets for therapy development. In this thesis I demonstrate that UPS perturbations are also present in other organ systems of severe ‘Taiwanese’ SMA mice and in other SMA models including intermediate Smn2B/− mice, zebrafish and patient derived iPSC motor neurons. Given the previously demonstrated improved neuromuscular phenotype in SMA mice treated with the β-catenin inhibitor quercetin I have been establishing whether other compounds with β-catenin inhibition offer similar or even better therapeutic options. Aspirin, indomethacin and iCRT-14 trials did not improve the SMA phenotype with likely off-target adverse effects meaning that quercetin remains the most tolerable β- catenin inhibitor in SMA mice to date. Another potential target of the UPS for SMA therapeutics is the deubiquitinating enzyme Uchl1, levels of which are increased in SMA. In this thesis I show that pharmacological inhibition of Uchl1 did not improve survival or motor performance in SMA mice and instead had a detrimental impact on the disease phenotype which could be explained by worsening SMA ubiquitin defects. Histological analysis revealed that there was no improvement in lower motor neuron count numbers, neuromuscular junction deficits or muscle fibre diameters. Mimicking the UPS phenotype in primary neuronal cells suggested that targeting UPS perturbations observed in SMA that are upstream of Uchl1, particularly the loss of Uba1, may therefore offer a more effective therapeutic option. Finally, I therefore examined whether increasing Uba1 levels in SMA mice using gene therapy technology was able to improve the SMA phenotype. My initial studies indicate that delivery of AAV9-UBA1 to SMA mice may be beneficial as intraperitoneal injection of AAV9-UBA1 was found to increase the weight and improve motor performance of SMA mice. Intravenous delivery of AAV9-UBA1 was found to further improve expression levels and biodistribution of AAV9-UBA1 in the central nervous system as well as systemically in all body organs and tissues. Western blot and proteomic analysis revealed that AAV9-UBA1 gene therapy is also able to correct downstream UPS perturbations found in SMA as well as increase SMN levels. Together, these results suggest that AAV9-UBA1 gene therapy is an exciting novel therapeutic approach for SMA.

Digitized by Kansas Correctional Industries

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder characterized by insufficient SMN protein, resulting in motoneuron death. Initially, it was thought that motoneuronal death is followed by muscle atrophy; however, recent research is beginning to reveal possible muscle intrinsic defects, independent of motoneuron defects, in SMA. Previous studies have elucidated the cooperative involvement of CARM1, HuD and SMN in motoneurons, revealing HuD as a possible key player in the SMA phenotype. In this study, we focus on HuR, a ubiquitous family member of HuD, and the possibility that it plays a similar key role with CARM1 and SMN in skeletal muscle. Through the use of an shCARM1 stable line of C2C12s, we show that CARM1 is necessary for HuR functionality during differentiation. We further show that the methylation of HuR is necessary for its capability to translocate cytoplasmically during differentiation. We confirm an interaction between HuR and SMN, suggestive of a similar mechanism as was shown previously with HuD. In light of these findings, we next progressed to determine whether HuR is misregulated in an SMA mouse model. We report increased CARM1 levels in skeletal muscles of these mice. We further discovered that a deficiency in SMN protein impairs HuR upregulation and cytoplasmic translocation in response to HuR activation through sciatic nerve denervation. These findings were correlated with aberrant mRNA expression of HuR targets upon denervation. Taken together, these results show that HuR methylation is essential for proper myogenesis, and that the mechanism by which it acts likely requires sufficient SMN protein levels. In a deficiency of SMN, HuR shows signs of misregulation that may play a role in the inability to maintain or repair muscle in SMA.