Proteomics-based identification and characterisation of spinal muscular atrophy disease pathways

Spinal muscular atrophy (SMA) is a debilitating genetic disorder, characterised by homozygous loss of the survival of motor neuron 1 (SMN1) gene, resulting in insufficient levels of ubiquitously expressed survival of motor neuron (SMN) protein. The traditional picture of SMA as a neuromuscular disease is slowly being changed by the findings of systemic pathology in SMA patients. This is also one of the reasons why therapeutic strategies aimed at increasing the levels of functional SMN protein are not completely effective, since they would often target neuromuscular pathology only. Here, the research was focused on identifying and characterising new disease pathways in SMA that could potentially be used as therapeutic targets in SMA. In the first results chapter, six proteins, lamin A/C, UBA1, ANXA2, GAPDH, NCAM and GAP43, identified previously in a multi-study comparison as having consistent direction of differential expression across three proteomic studies of SMA, were investigated in SMA tissues and cells using western blotting. Of these, lamin A/C, UBA1, ANXA2 and GAPDH showed widespread dysregulation across a range of tissues from severe Taiwanese mouse model of SMA and in fibroblast cells from SMA patients, suggesting that each one of these proteins might have a role in SMA disease pathways. Selection of drugs that can target these proteins proved to be very challenging, and further efforts are needed to identify appropriate therapeutic strategies. In the second results chapter, the potential role for lamin A/C in SMA disease pathways was further highlighted by findings of mechanistic link between lamin A/C and UBA1, a known SMA modifier. Lamin A/C dysregulation might be especially relevant in the context of heart pathology, where increased levels of lamin A/C would likely stiffen the cardiomyocytes and impair normal heart function. In the third results chapter, multi-study comparison of published proteomic studies of amyotrophic lateral sclerosis (ALS) identified core protein changes in ALS tissues and cells, and biochemical investigation of two proteins, ALDOA and GAPDH, highlighted defects in cell metabolism as an important disease mechanism in ALS. Of these, fifteen proteins were also differentially expressed across at least two proteomic studies of SMA, however, when investigated biochemically in SMA and ALS mouse spinal cords, these proteins showed very little evidence that two diseases converge on the same molecular mechanisms. When taken together, results presented here show that published proteomic studies contain a wealth of information that often get overlooked when examined in isolation. These datasets were exploited here to identify core proteins and molecular mechanisms that drive disease pathogenesis in SMA and ALS. Knowledge of general disease mechanisms may allow development of therapies that can systemically target peripheral pathology in SMA. For example, systemic restoration of lamin A/C levels might prove useful for correcting defects in a range of tissues, with an emphasis on stiff tissues like the heart and muscles, and when combined with an SMN-targeted approach, it might bring greater therapeutic benefit to SMA patients compared to SMN-targeted approach alone. In conclusion, several proteins identified in a multi-study comparison, including lamin A/C, GAPDH and ALDOA, were linked to different SMA and ALS disease pathways, providing evidence that multi-study comparison has the power to identify core disease-related protein changes. This thesis also opened a range of new questions that need to be addressed in the future work, including the selection of drugs that can restore defects in the expression or activity or target proteins. Many other proteins, identified in multi-study comparisons, could not be investigated here in more detail. These proteins may help to further expand the knowledge of SMA and ALS disease pathways and therefore demand further experimental attention