Theses - MRC Toxicology Unit
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Item Embargo TAp73 regulates mitochondrial dynamics through an OPA1 axisBuckley, NiallMitochondria are energy-producing organelles. They are highly adaptive and undergo the processes of fusion and fission to couple mitochondrial function with changing cellular demands. In this thesis, I have identified a new molecular mechanism involving TAp73/OPA1 that controls mitochondrial morphology (Buckley et al., 2020). OPA1 drives fusion of the inner mitochondrial membrane and controls cristae remodelling, a process facilitating the execution of apoptosis. I have shown that TAp73 regulates OPA1 expression in TAp73-/- cell lines which were generated using CRISPR/Cas9 targeting. Concurrently, I show that disruption of this TAp73/OPA1 axis results in a fragmented mitochondrial network owing to impaired mitochondrial fusion. Disruption of this axis also reduces the capacity for TAp73-/- cells to produce energy via oxidative phosphorylation. Further, the ectopic expression of OPA1 in TAp73-/- cells rescued defective mitochondria and restored bioenergetic function, placing OPA1 downstream of TAp73 in the regulation of mitochondrial dynamics. Decreased expression of OPA1 also results in altered cristae structure in cellular and in vivo models with deletion of TAp73. Importantly, owing to the role of OPA1 in modulating cytochrome c release, TAp73-/- cells have an increased sensitivity to apoptotic cell death, e.g., on exposure to BH3-mimetics. Further, many cancers such as lung and colon have upregulated expression of TAp73 which is associated with poor survival outcomes. This raises the possibility that the TAp73/OPA1 axis may be hijacked in cancer to evade apoptotic cell death and increase energy production, thereby facilitating tumorigenesis and supporting a growth-promoting role of TAp73 isoforms. This TAp73/OPA1 axis is also important in the respiratory system, owing to high levels of TAp73 expression in airway epithelial cells. Indeed, the correct ciliation of airway cells is severely perturbed in TAp73 null mice. I therefore propose that these profound defects in ciliogenesis may be driven by dysregulated mitochondrial function. This was highlighted by the fact that the airway epithelium of p73 null mice displayed downregulation of OPA1 and an altered morphology of the mitochondrial network in the ciliated cell lineage. Further, defective cilia have a reduced capacity for mucociliary clearance of inhaled toxic particles. Loss of TAp73 function and the concomitant increase in sensitivity to apoptosis may therefore further enhance vulnerability to inhaled pathogens or pollutants, highlighting a possible role for TAp73 in the incidence and progression of airway diseases such as COPD.Item Open Access The role of organelle crosstalk in a Drosophila model of Parkinson’s diseasePopovic, Rebeka; Popovic, Rebeka [0000-0002-6839-9391]Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by the loss of dopaminergic (DA) neurons, causing deficits in motor function. At present, mitochondrial dysfunction and the endoplasmic reticulum (ER) unfolded protein response (UPR) are perceived to be molecular features of PD. The UPR is an evolutionarily conserved adaptive cellular response to unfolded or misfolded proteins in the ER. Three ER-resident proteins sense the UPR, one of which is the protein kinase RNA (PKR)-like ER kinase (PERK). PERK activation leads to translational repression via phosphorylation of eukaryotic translation initiation factor-2 α (eIF2α) as well as activation of activating transcription factor 4 (ATF4). This triggers a transcriptional response, initially promoting cell survival. Eventually, the sustained activation of the UPR leads to cell death. PERK has also been reported to modulate mitochondrial function. The overarching aim of this thesis was to investigate the relationship between PERK kinase and mitochondrial dysfunction in the pathogenesis of PD using the fruit fly Drosophila melanogaster as a model organism. To characterise the Drosophila PERK (dPerk) expressional landscape, I combined microarray and quantitative proteomics analysis from adult flies overexpressing dPerk. Using this approach, I identified tribbles (trbl) and Heat shock protein 22 (Hsp22) as two novel Drosophila ATF4 (dAtf4) regulated transcripts. Furthermore, I established that dPerk expression leads to translational repression of several mitochondrial proteins. The mitochondria-ER tether ATPase Family AAA Domain Containing 3A (ATAD3A) has recently been suggested to function as a negative regulator of PERK in mammalian models. I show that the ATAD3A fly orthologue Belphegor (Bor) does not act as an inhibitor of dPerk. Furthermore, I propose that this is due to the lack of the proline-rich motif in Drosophila, otherwise present in the protein structure of its mammalian orthologue. Mutations in PTEN-induced putative kinase 1 (PINK1), a mitophagy gene, cause neurodegeneration in some autosomal recessive forms of PD. In Drosophila, mutations in the pink1 gene cause mitochondrial dysfunction and the degeneration of DA neurons. Pink1 mutants also show overactivation of the dPerk/eIF2α/dAtf4 axis. Therefore, I used the pink1 PD Drosophila model to further probe the role of dPerk in the pathomechanism of PD. PD patients experience gastrointestinal issues that often precede the onset of motor symptoms, implicating the gut-brain axis in the pathogenesis of this disease. Likewise, in pink1 mutants, mitochondrial dysfunction leads to cell death and proliferation in the Drosophila midgut. Suppressing intestinal dysfunction is neuroprotective. I found that the intestinal expression of dPerk causes intestinal damage, and silencing dPerk in the pink1 intestine leads to a rescue of intestinal dysfunction and neurodegeneration. The ER stress marker Trbl also acts as an inhibitor of Akt, implicating this pseudokinase as a negative regulator of insulin signalling. The human Trbl orthologue tribbles pseudokinase 3 (TRIB3) is upregulated in insulin resistant as well as obese adults, and TRIB3 polymorphisms are associated with type 2 diabetes and insulin resistance. The fat body is a fly organ homologous to the mammalian liver and adipose tissue. It functions to synthesise and store triacylglycerol and regulate endocrine and immune signalling, implicating it in the regulation of complex behaviours such as sleep. My results show that the FB expression of Trbl leads to metabolic defects, systemic repression of insulin signalling and a reduction in night-time sleep. This thesis investigates inter-organelle as well as inter-organ signalling using Drosophila melanogaster as a model organism. My research shows that the ER UPR kinase dPerk is an important regulator of mitochondrial function and intestinal homeostasis, suggesting dPerk as a potential therapeutic target for PD. Furthermore, analysis of Trbl pseudokinase function in Drosophila melanogaster proposes Trbl as a molecular link between animal nutrient state and behaviour.Item Open Access In vivo and in vitro approaches to characterising axon growth and metabolic alterations in Parkinson’s diseaseTravaglio, MarcoA recent pathological hypothesis of Parkinson’s disease suggests that at the time of disease onset, midbrain dopaminergic cells lose their synaptic connectivity before dying. The preposition that dopaminergic axons degenerate initially and predominantly in PD offers new critical opportunities for the development of neuroprotective and restorative therapies. However, we know little about how dopaminergic neurons innervate their target areas. Because of their functional importance in PD, understanding the mechanisms underlying the development and function of midbrain dopaminergic circuits is of considerable interest. Therefore, the initial focus of my research was on exploring novel pathways governing the development of dopaminergic circuitry. Using a mouse model, my work defined a novel role for Nolz1, a developmentally expressed transcription factor, in regulating aspects of dopaminergic axon growth and target innervation. I show that Nolz1 regulates critical aspects of striatal development in the mouse embryo and that aberrant striatal patterning alters the outgrowth of nigrostriatal projection neurons (Chapter 2). The premature degeneration of axonal projections in PD has been linked to changes in mitochondrial function. Impairment of mitochondrial dynamics, transport, and loss of plasticity of axon terminals precedes the onset of neuronal degeneration in this neurodegenerative disease. PINK1 is a mitochondrial kinase that ensures mitochondrial health and mutations in PINK1 cause PD. My work explored how mutations in PINK1 alter cellular metabolism, using a Drosophila model of PD. Multi-omics analysis of pink1 mutant flies uncovered metabolic and transcriptional modifications in cysteine metabolism which coincided with defects in mitochondrial respiration. These findings confirmed PINK1’s canonical role in mitochondrial function, while highlighting the relevance of cysteine metabolism to compensate for early and selective defects in mitochondrial respiration and ATP production (Chapter 3). To answer if we can recapitulate these changes in a human model, I then used human induced pluripotent stem cells (iPSCs). I differentiated human induced pluripotent stem cells (iPSCs) carrying a recently discovered I368N mutation in PINK1 into neural precursor cells (NPCs) and examined their metabolic profile. I found that the PINK1 I368N mutation causes global metabolic changes that broadly validate the comprehensive number of hits recovered in pink1 mutant flies, suggesting a significant overlap between my integrated networks. This convergence of disease phenotypes points to a common disease mechanism that may offer a unifying perspective on early-stage PD pathology (Chapter 4). Together, these results shed light on novel genes and pathways that could be exploited for therapeutic intervention.