AbstractDuchenne muscular dystrophy (DMD) is the most common inherited muscle disease in men and currently there is no effective treatment for this debilitating and lethal disorder. Although the absence of dystrophin is identified as the main cause of DMD, multiple secondary changes have been found to result from the dystrophin deficiency both in muscle and in non-muscle tissues. Among these abnormalities, our laboratory and others have demonstrated a dramatic increase in the expression of the P2RX7 receptor in cells and tissues from DMD patients and the mdx mouse model of DMD.
The aim of this study was to determine the effects of P2RX7 ablation on biological functions in mdx muscle in situ and to identify suitable diagnostic and prognostic biomarkers for use in studies on the pharmacological inhibition of P2RX7. RNA transcription was profiled in muscle from wild-type and mdx mice, and also from double knock-out mdx/P2rx7-/- mice which lack both functional Dmd and P2rx7 genes, and the effects of P2RX7 antagonists were assessed on pathological markers at the acute disease stage of disease in mdx mice, which resembles the human pathology.
RNA sequencing was performed using the Illumina HiSeq 2000 platform to characterise the differential gene expression in tibialis anterior (TA) muscles from four week old wild type, mdx and mdx/P2rx7-/- mice. The biological functions and molecules that are most affected in mdx and corrected in mdx/P2rx7-/- tissues are those of the immune response, including the innate immune response, cytokine regulatory genes and the NF-кB pathway followed by fibrosis, telomerase regulatory genes, atherosclerosis signalling and the LXR/RXR activation pathway. Moreover, activation of the cell cycle, mitochondrial dysfunction, apoptosis and the adherens junction genes were found to be altered in mdx compared to wild type but not normalised in mdx/P2rx7-/- muscles. This analysis also demonstrated that the mdx mutation disrupts the non-sense-mediated RNA decay and splicing mechanisms, which leads to an increase in out-of-frame transcripts that may have unexpected cellular impact. One of these altered transcripts, Bmp7, was significantly down-regulated in the mdx myoblasts, myofibres and in TA muscles and restored to the normal level in mdx/P2rx7-/- muscle. Different analyses were used to map this alteration to the 5'exons of Bmp7 but the identification of this abnormal transcript was not successful.
Four P2RX7 antagonists (oxidised ATP, A438079, AFC-5128 and azidothymidine (AZT)) were administered to male mdx mice and their effects on the pathology were analysed using different methods and biomarkers. All of the antagonists inhibited P2RX7 receptor-mediated responses in mdx mice without any detectable side effects. A438079 and AZT were found as the most effective in attenuating the wide range of the pathological features.
The reduction in dystrophic features as results of ablation and antagonism the P2RX7 confirms the involvement of this purinoreceptor in the DMD pathology and making it an attractive target for a pharmacological treatment of this lethal disease. Additionally, as AZT is already in clinical use for other diseases, also in children, this drug could be relatively easily re-purposed and trialled for the treatment of DMD.
|Date of Award||Mar 2017|
|Supervisor||Darek Gorecki (Supervisor) & Stephen Arkle (Supervisor)|