Abstract
Spinal muscular atrophy (SMA) is an autosomal recessive childhood motor neuron disease, and the most common genetic cause of infant mortality worldwide. It is characterised by the loss of the survival motor neuron (SMN) protein, leading to motor neuron degeneration and progressive muscle wasting. Substantial medical advancements have taken place since 2016 with the approval of the first therapy for SMA, with a number following shortly after. These therapies all share a common pathway: restoration of SMN. Despite this, some challenges remain, as crucially, not all patients respond to SMN-enhancing treatments. Moreover, the concept of SMA as solely a disease of the motor neurons is outdated, shifting instead towards the definition of a ‘whole-body’ disorder, as evidence grows for the contribution of other tissues to the overall disease phenotype. The influence of non-neuronal tissue and systemic pathologies in SMA development represents an important consideration when developing novel therapeutics.The variability of disease modifiers contributing to the broad phenotypic variability in the SMA phenotype may also contribute to treatment response. A number of genes encoding the proteins with a role in actin dynamics and related processes have been reported as SMA modifying factors. DNA methylation, a common epigenetic modification, can be a mediator or modifier in many common diseases. A wholegenome methylation pattern analysis reported significant decrease in methylation of the SPECC1 gene in SMA patients compared with healthy controls. This gene and corresponding CYTSB protein have been associated with SMA pathogenesis-related processes such as apoptosis, ribosomal biogenesis and metabolism, and has potential for actin binding.
This study describes novel characterisations of the SPECC1 gene and CYTSB protein, both in physiological conditions, and in the context of SMA pathophysiology. Using bioinformatic analysis and modelling, several domains were identified and characterised including the actin binding CH-domain which varied slightly among the four key isoforms. Homologous sequences were identified both at the gene and protein level, and also common potential protein-protein interactions were found, namely with relation to growth and maintenance functions within the cell. Bioinformatics and immunofluorescence analyses found CYTSB located in both the nucleus and cytoplasm of the cell. Furthermore, molecular analyses of SPECC1 mRNA and CYTSB protein demonstrated altered expression across brain, heart, liver, kidneys, spinal cord and gastrocnemius muscle in both healthy and SMA mice.
In SMA mice, the different isoforms of CYTSB demonstrated tissue-specific and timespecific expression. Isoform 3 was only present in mouse liver at P0, regardless of genotype. Similarly, expression of isoforms 1 and 4 were found in SMA mice gastrocnemius muscles and liver tissue and, respectively, at P8 but not in their agematched controls, further signifying potential tissue-specific or development-specific roles for the specific isoforms.
In SMA-like HEK293 cells produced through siRNA knockdown experiments, SPECC1 mRNA was increased. Conversely, when SPECC1 was knocked down in HEK293 cells, there was no downstream effect on SMN. There was also no relationship identified between UBA1 and SPECC1 or SMN, although there was also a trend towards an increase in UBA1 in SMN-deficient cells.
CYTSB isoforms 1, 3 and 4 were successfully cloned and overexpressed in HEK293 cells through Gibson cloning. Co-immunoprecipitation experiments and apoptosis assays were conducted following transfection with the overexpression constructs; however, results of these experiments were inconclusive.
In conclusion, there are a number of connections between SPECC1/CYTSB and SMA via key cellular processes such as apoptosis, actin dynamics, and cellular signalling and trafficking. This work shines light on the potential of SMN-adjacent or - independent pathways as a key therapeutic pathway to consider for future SMA therapies.
| Date of Award | 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Gillian Hunter (Supervisor) & Fiona Kerr (Supervisor) |