Cell lineage-specific mitochondrial resilience during mammalian organogenesis

Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments

Mechanisms Controlling the Segregation of Mitochondrial DNA Heteroplasmy

Mutations of the mitochondrial DNA (mtDNA) are often the cause behind primary mitochondrial disorders affecting 1:5000 individuals. However, the full extent of the impact that mtDNA mutations have is yet to be comprehensively understood. One of the main reasons behind our slow progress in the field is the multi-copied nature of mtDNA, which suggests that even healthy individuals will carry a small percentage of mutated mtDNA molecules alongside healthy ones, in a state termed heteroplasmy. In cases where the proportion of mutant to healthy mtDNA molecules reaches a critical threshold, diverse and multisystem pathological phenotypes begin to appear. While an individual’s mtDNA heteroplasmy level is largely dependent on that of his maternal germline, studies have shown that there are diverse forces, both intra and extracellular in nature that drive segregation. Further complicating this phenomenon, the observed driving forces appear to be mutation- and cell type-specific in their effect. In this dissertation I first describe my work on optimising and validating a protocol that allows us to measure single cell heteroplasmy. Developing this in-house technique, enabled us to perform high-throughput analyses of cell populations of interest while revealing for the first time the intricacies governing single mtDNA heteroplasmy variability at the single cell level. With this protocol in place, I set out to study the heteroplasmy of mouse brain- and spleen-derived populations. In this endeavour, I made use of two novel mouse models that carry a mutation on mitochondrial-tRNA Alanine (mt-Ta), m.5019A>G and m.5024C>T. Recording single cell heteroplasmy values at different timepoints throughout development, we observed that both mutations followed the principles of random genetic drift. The rate of drift exhibited mutation-specific patterns. Moreover, I present a collaborative project geared towards uncovering the impact the two mt-Ta mutations have at the level of the transcriptome on difference cell lineages belonging to E8.5 mouse embryos. I describe the identification of 17 distinct cell lineages and their inherent variability in mtDNA transcript abundance. While no developmental disparities were observed in mutant embryos compared to controls, we did detect an upregulation of mtDNA transcripts in response to the mutation. At the same time, genes that were previously defined as epistatic suppressors/buffers were found to be downregulated. Pseudobulk analysis revealed differential expression of genes both at the level of the organism and that of the cell-lineage. Overall, mice carrying the m.5024C>T mutation seem to mount a greater compensatory transcriptional response compared to their m.5019A>G counterparts. Finally, I explore the relationship between mtDNA heteroplasmy, copy number and the cell cycle. More specifically, making use of a fluorescent cell cycle reporter, I examine mtDNA changes along the cell cycle. Having established a consistent pattern, I assess the impact of genetic manipulation of mtDNA copy number and restriction of glycolysis on cell cycle progression. Finally, I delve into the consequences of large scale mtDNA deletions on the cell’s respiratory capacity and examine whether that defect impacts their ability to complete the cell cycle.Work carried out as part of this PhD, was funded by the UK Medical Research Council

Cell-Free Mitochondrial DNA in Acute Brain Injury.

Traumatic brain injury and aneurysmal subarachnoid haemorrhage are a major cause of morbidity and mortality worldwide. Treatment options remain limited and are hampered by our understanding of the cellular and molecular mechanisms, including the inflammatory response observed in the brain. Mitochondrial DNA (mtDNA) has been shown to activate an innate inflammatory response by acting as a damage-associated molecular pattern (DAMP). Here, we show raised circulating cell-free (ccf) mtDNA levels in both cerebrospinal fluid (CSF) and serum within 48 h of brain injury. CSF ccf-mtDNA levels correlated with clinical severity and the interleukin-6 cytokine response. These findings support the use of ccf-mtDNA as a biomarker after acute brain injury linked to the inflammatory disease mechanism

Intracellular Chloride Channels Regulate Endothelial Metabolic Reprogramming in Pulmonary Arterial Hypertension

Mitochondrial fission and a metabolic switch from oxidative phosphorylation to glycolysis are key features of vascular pathology in pulmonary arterial hypertension (PAH) and are associated with exuberant endothelial proliferation and apoptosis. The underlying mechanisms are poorly understood. We describe the contribution of two intracellular chloride channel proteins, CLIC1 and CLIC4, both highly expressed in PAH and cancer, to mitochondrial dysfunction and energy metabolism in PAH endothelium. Pathological overexpression of CLIC proteins induces mitochondrial fragmentation, inhibits mitochondrial cristae formation, and induces metabolic shift toward glycolysis in human pulmonary artery endothelial cells, consistent with changes observed in patient-derived cells. Interactions of CLIC proteins with structural components of the inner mitochondrial membrane offer mechanistic insights. Endothelial CLIC4 excision and mitofusin 2 supplementation have protective effects in human PAH cells and preclinical PAH. This study is the first to demonstrate the key role of endothelial intracellular chloride channels in the regulation of mitochondrial structure, biogenesis, and metabolic reprogramming in expression of the PAH phenotype

Cell lineage-specific mitochondrial resilience during mammalian organogenesis.

Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments