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Modelling neuronal mitochondrial aminoacyl-tRNA synthetase defects https://doi.org/10.17863/CAM.118324 Podmanicky, Oliver Horvath, Rita Mitochondrial diseases cover a broad group of disorders caused by mitochondrial dysfunction, often affecting organs with high metabolic demand, such as the brain and skeletal muscle. Mutations in mitochondrial aminoacyl-tRNA synthetase (MT-ARS) genes, which are crucial for mitochondrial protein synthesis and energy production via oxidative phosphorylation, are implicated in a variety of severe neurological and multisystemic diseases. Among these, mutations in mitochondrial alanyl-tRNA synthetase (AARS2), mitochondrial glutamyl-tRNA synthetase (EARS2), and mitochondrial arginyl-tRNA synthetase (RARS2) cause distinct clinical manifestations including combined oxidative phosphorylation deficiency type 8 (COXPD8), characterised by leukoencephalopathy with ovarian failure, or cardiomyopathy, leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL), and pontocerebellar hypoplasia type 6 (PCH6). Despite their significance, the underlying pathological mechanisms remain poorly understood due to the lack of physiologically relevant human models, as current research relies primarily on non-human models or patient-derived fibroblasts that do not recapitulate the tissue-specific complexities of the nervous system. Here, I address this unmet need by applying tissue-specific human models of the nervous system to investigate the cellular impact of AARS2, EARS2 and RARS2 defects. First, patient fibroblasts were reprogrammed into neuronal cells, which allows for a direct comparison of different MT-ARS mutations. These in vitro models exhibit the tissue-specificity of MT-ARS mutations by showing selective loss of mitochondrial respiratory chain complexes in differentiated neurons but not in proliferating neural progenitor cells. RNA sequencing, immunoblotting and mitochondrial respiratory analysis of these models demonstrate varying degrees of mitochondrial dysfunction along with the activation of distinct compensatory mechanisms and cellular stress responses, including the integrated stress response, and impairment in neuronal development. Furthermore, I investigate how AARS2 mutations associated with two distinct phenotypes impact mitochondrial protein synthesis in neurons in vitro using a non-radioactive technique involving click chemistry. In addition to developing neuronal models of MT-ARS defects, I sought to complement the in vitro findings with a viable in vivo model of EARS2 deficiency. Given the housekeeping role of MT-ARS, homozygous mutations in these genes are often embryonically lethal and in vivo models are rarely viable. Zebrafish offer a unique advantage in this regard, enabling viable MT-ARS whole-body knockouts to investigate developmental and systemic consequences of these mutations. I present new findings on the pathogenicity of EARS2 mutations in early development and test potential therapeutic strategies for MT-ARS-related disorders in vivo using a transgenic zebrafish model. Ears2 knockout zebrafish presented with abnormal development, early lethality, reduced locomotor activity, and activation of cellular stress, which was partially rescued by amino acid supplementation. University of Cambridge 2024-09-29 Thesis Doctoral eng https://www.repository.cam.ac.uk/handle/1810/384272 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a31af9a8-6cb0-4d16-8f91-f73a796c7b15/download b6d236bf6ee8892018bdc13753ee624a https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/920d6fef-7fd5-4e21-95a8-49d6ca4f4759/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved aminoacyl-trna synthetase mitochondria translation AARS2 EARS2 RARS2