Spatial and subcellular gene expression patterns in myogenic differentiation and muscle pathology
<p>Duchenne muscular dystrophy (DMD) is a debilitating muscle-wasting disorder caused by lack of dystrophin protein. Promising dystrophin-restoration therapies such as gene editing using the CRISPR-Cas9 system and antisense oligonucleotide-mediated exon skipping using peptide-phosphorodiamidat...
| Main Author: | |
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2024
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| _version_ | 1824458859675123712 |
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| author | Chwalenia, KM |
| author2 | Roberts, T |
| author_facet | Roberts, T Chwalenia, KM |
| author_sort | Chwalenia, KM |
| collection | OXFORD |
| description | <p>Duchenne muscular dystrophy (DMD) is a debilitating muscle-wasting disorder caused by lack of dystrophin protein. Promising dystrophin-restoration therapies such as gene editing using the CRISPR-Cas9 system and antisense oligonucleotide-mediated exon skipping using peptide-phosphorodiamidate morpholino oligomers (PPMOs) are currently in development. The efficacy of these approaches is typically assessed in terms of the amount of dystrophin restored, while correct sarcolemmal localisation of the protein has largely been assumed.</p>
<p>Our prior work revealed that CRISPR-Cas9-mediated exon excision results in a spatially restricted, patchy pattern of dystrophin expression. In this thesis, a novel DMD mouse model (<em>mdx52-XistΔhs</em>) was developed, characterised by chimeric myofibres, consisting of both dystrophin-expressing and non-dystrophin-expressing nuclei as a consequence of skewed X-chromosome inactivation. Non-uniform dystrophin localisation along myofibres of <em>mdx52-XistΔhs</em> animals mirrors the pattern observed in CRISPR-Cas9-treated animals. These observations are consistent with the myonuclear domain theory which posits that every nucleus in syncytial myofiber controls a specific amount of cytoplasm around it. Analysis of centrally nucleated myofibres as well as serum myomiR biomarkers (miR-1, miR-133 and miR-206) reveals that patchy dystrophin positively modifies but does not prevent the muscle degeneration. Moreover, disorganisation of microtubule network in dystrophic mice is only partially rescued by the presence of dystrophin positive myonuclear domains. Additionally, visualisation of aged <em>mdx52-XistΔhs</em> muscle shows that the non-uniform pattern of dystrophin distribution and myofibre central nucleation do not resolve with age. Furthermore, single myofibre analysis reveals the presence of distinct myofibre classes in <em>mdx52-XistΔhs</em> muscle differentiated by the position of their respective myonuclei. Systematic classification of individual <em>mdx52-XistΔhs</em> myofibres shows profound differences associated with central nucleation. These include higher numbers of myonuclei and the more substantial disorganisation of the microtubule network in centrally nucleated myofibres. Intriguingly, analysis of dystrophin expression in centrally nucleated regions of <em>mdx52-XistΔhs</em> myofibres reveals that dystrophin is translationally repressed in these areas.</p>
<p>In contrast to non-uniform dystrophin expression in <em>mdx52-XistΔhs</em> and CRISPR-Cas9 treated mice, treatment with PPMO exon skipping compound restores dystrophin uniformly at the sarcolemma in <em>mdx</em> dystrophic animals. Notably, this uniform pattern is observed regardless of the PPMO dose. Additionally, exon skipping, and levels of dystrophin protein rescue are inversely correlated with serum myomiRs abundance, emphasising their utility as pharmacodynamic markers for dystrophin restoration strategies.</p>
<p>Within the muscle tissue myomiRs are known to regulate the process of myogenesis which supports skeletal muscle turnover in DMD. Canonically, microRNAs have been considered to function only in the cytoplasm. However, analyses in murine and human muscle cells shows that myomiRs are abundant and specifically upregulated in the nucleus during myogenic differentiation. Moreover, the majority of myomiRs localise to the nucleoplasm and are bound to the effector AGO2 protein. Accordingly, AGO2 protein localises primarily to the nucleus of the differentiating mouse muscle cells. These results reveal a previously unappreciated aspect of myomiRs function during muscle formation.</p> |
| first_indexed | 2025-02-19T04:32:35Z |
| format | Thesis |
| id | oxford-uuid:102f8022-3503-480a-ad8c-cbf43dcb1c22 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2025-02-19T04:32:35Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | oxford-uuid:102f8022-3503-480a-ad8c-cbf43dcb1c222025-01-16T06:34:41ZSpatial and subcellular gene expression patterns in myogenic differentiation and muscle pathologyThesishttp://purl.org/coar/resource_type/c_db06uuid:102f8022-3503-480a-ad8c-cbf43dcb1c22EnglishHyrax Deposit2024Chwalenia, KMRoberts, T<p>Duchenne muscular dystrophy (DMD) is a debilitating muscle-wasting disorder caused by lack of dystrophin protein. Promising dystrophin-restoration therapies such as gene editing using the CRISPR-Cas9 system and antisense oligonucleotide-mediated exon skipping using peptide-phosphorodiamidate morpholino oligomers (PPMOs) are currently in development. The efficacy of these approaches is typically assessed in terms of the amount of dystrophin restored, while correct sarcolemmal localisation of the protein has largely been assumed.</p> <p>Our prior work revealed that CRISPR-Cas9-mediated exon excision results in a spatially restricted, patchy pattern of dystrophin expression. In this thesis, a novel DMD mouse model (<em>mdx52-XistΔhs</em>) was developed, characterised by chimeric myofibres, consisting of both dystrophin-expressing and non-dystrophin-expressing nuclei as a consequence of skewed X-chromosome inactivation. Non-uniform dystrophin localisation along myofibres of <em>mdx52-XistΔhs</em> animals mirrors the pattern observed in CRISPR-Cas9-treated animals. These observations are consistent with the myonuclear domain theory which posits that every nucleus in syncytial myofiber controls a specific amount of cytoplasm around it. Analysis of centrally nucleated myofibres as well as serum myomiR biomarkers (miR-1, miR-133 and miR-206) reveals that patchy dystrophin positively modifies but does not prevent the muscle degeneration. Moreover, disorganisation of microtubule network in dystrophic mice is only partially rescued by the presence of dystrophin positive myonuclear domains. Additionally, visualisation of aged <em>mdx52-XistΔhs</em> muscle shows that the non-uniform pattern of dystrophin distribution and myofibre central nucleation do not resolve with age. Furthermore, single myofibre analysis reveals the presence of distinct myofibre classes in <em>mdx52-XistΔhs</em> muscle differentiated by the position of their respective myonuclei. Systematic classification of individual <em>mdx52-XistΔhs</em> myofibres shows profound differences associated with central nucleation. These include higher numbers of myonuclei and the more substantial disorganisation of the microtubule network in centrally nucleated myofibres. Intriguingly, analysis of dystrophin expression in centrally nucleated regions of <em>mdx52-XistΔhs</em> myofibres reveals that dystrophin is translationally repressed in these areas.</p> <p>In contrast to non-uniform dystrophin expression in <em>mdx52-XistΔhs</em> and CRISPR-Cas9 treated mice, treatment with PPMO exon skipping compound restores dystrophin uniformly at the sarcolemma in <em>mdx</em> dystrophic animals. Notably, this uniform pattern is observed regardless of the PPMO dose. Additionally, exon skipping, and levels of dystrophin protein rescue are inversely correlated with serum myomiRs abundance, emphasising their utility as pharmacodynamic markers for dystrophin restoration strategies.</p> <p>Within the muscle tissue myomiRs are known to regulate the process of myogenesis which supports skeletal muscle turnover in DMD. Canonically, microRNAs have been considered to function only in the cytoplasm. However, analyses in murine and human muscle cells shows that myomiRs are abundant and specifically upregulated in the nucleus during myogenic differentiation. Moreover, the majority of myomiRs localise to the nucleoplasm and are bound to the effector AGO2 protein. Accordingly, AGO2 protein localises primarily to the nucleus of the differentiating mouse muscle cells. These results reveal a previously unappreciated aspect of myomiRs function during muscle formation.</p> |
| spellingShingle | Chwalenia, KM Spatial and subcellular gene expression patterns in myogenic differentiation and muscle pathology |
| title | Spatial and subcellular gene expression patterns in myogenic differentiation and muscle pathology |
| title_full | Spatial and subcellular gene expression patterns in myogenic differentiation and muscle pathology |
| title_fullStr | Spatial and subcellular gene expression patterns in myogenic differentiation and muscle pathology |
| title_full_unstemmed | Spatial and subcellular gene expression patterns in myogenic differentiation and muscle pathology |
| title_short | Spatial and subcellular gene expression patterns in myogenic differentiation and muscle pathology |
| title_sort | spatial and subcellular gene expression patterns in myogenic differentiation and muscle pathology |
| work_keys_str_mv | AT chwaleniakm spatialandsubcellulargeneexpressionpatternsinmyogenicdifferentiationandmusclepathology |