Ellwood, Rebecca A.
(2022)
Effects of compounds on C. elegans DMD model health.
PhD thesis, University of Nottingham.
Abstract
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin gene. The dystrophin gene encodes a cytoskeletal protein with the same name that is responsible for ensuring the strength, stability, and functionality of myofibers. In DMD the dystrophin protein is either absent or there are insufficient levels of functional dystrophin resulting in progressive muscular damage and degeneration. This results in muscular weakness, motor delays, loss of ambulation, and a shortened life expectancy due to respiratory impairment and cardiomyopathy. Treatment options are limited and mainly focused on alleviating symptoms; they are not a cure. The main therapeutic treatment used are glucocorticoids, these can be used for a couple of years, but treatment is often ceased due to undesirable side effects. An emerging therapy is the use of exon-skipping but currently these can only be used for patients amenable to skipping of exons 45, 51, and 53 (approximately 30% of patients). There is therefore a great need for alternative therapies.
This thesis uses C. elegans as a model for DMD. We demonstrate throughout that the C. elegans DMD model has clinical relevance as it shares some of the underlying pathophysiology that are also displayed in patients, including mitochondrial dysfunction and calcium dysregulation. It also has several clinically relevant phenotypes that can be exploited including movement and strength decline and changes in gait. Finally, the current standard treatment used to treat patients with DMD, prednisone, has also been identified as being beneficial in the DMD C. elegans model as well.
Hydrogen sulfide (H2S) compounds were trialled as a potential treatment for DMD in this thesis. The rationale behind this was that H2S compounds had been demonstrated previously to improve lifespan in ageing animals. There are some similarities between ageing and DMD muscle, the former being associated with sarcopenia and the latter with progressive muscle degeneration. It therefore seemed reasonable to expect an improvement in the DMD animals given there was one in ageing animals. In Chapter 3, we started by trialling a non-targeted H2S compound sodium GYY4137 (NaGYY) and showed that this compound does improve movement, strength, and gait in the DMD model. The basis of this improvement was likely mitochondrial, and the mechanism of action was like that of prednisone.
In Chapter 4, to confirm the basis of this we then used a mitochondrially targeted H2S compound, AP39, and demonstrated that this compound was also able to improve movement and strength in DMD animals. This provided further evidence to suggest that at least part of the mechanism of NaGYY was through improvements in mitochondrial dysfunction. We then further probed the mechanism of action of AP39 in the mitochondria and established that AP39 is likely donating electrons to complex III of the electron transport chain (ETC) and thus causing an increase in ATP content.
In Chapter 5, we show that there is a decline in the gene expression of enzymes responsible for sulfur metabolism that are resulting in a H2S deficit. We also demonstrate that supplementing sulfur in a different way (via sulfur containing amino acids) is also beneficial in the C. elegans DMD model. This highlights a potential novel underlying pathophysiology of DMD in a defective sulfur metabolism pathway and the potential of using H2S as a biomarker for disease progression.
To conclude we have shown that supplementing H2S compounds and sulfur containing amino acids are potential treatments for DMD and potential alternatives to prednisone. We have also demonstrated that manipulation of the sulfur metabolism pathway warrants further study in DMD. Future work includes trialling these therapies in the DMD mouse model and beyond and identifying whether the defective sulfur metabolism pathway and H2S deficit corresponds with higher organisms.
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