Theses - MRC Toxicology Unit
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Item Controlled Access A novel antigen-receptor signalling reporter mouse reveals clonotype-independent intratumoral trapping as a spatial feature of CD8+ T cell exhaustionSo, TszInterrogation of multi-clonal antigen-specific populations of lymphocytes can reveal fundamental principles about lymphocyte behaviour, but this has been difficult to achieve with existing technologies. A precise *in vivo* tool is required not only for the unbiased selection of antigen-specific lymphocytes against target antigens in an immune reaction, but also for the defined time-controlled sampling of their time-dependent fates. We have developed a novel mouse model, termed Antigen-Receptor Signalling Reporter (AgRSR) mouse, that uses the specificity of Nur77 (NR4A1) for antigen receptor activation to drive expression of a tamoxifen-inducible CreERT2, enabling time-stamping of TCR-activated lymphocytes with EYFP expression through a Cre-LoxP mechanism. This allows multiple clonal lymphocytes and their progeny to be permanently labelled. Combining isolation of the EYFP+ cells with further technologies would enable in-depth analysis of their cell states, clonotype, tissue location, and labelling localization. Validating the AgRSR model, I first showed that the system is robust in its dependencies for TCR signalling and tamoxifen administration for EYFP induction in T lymphocytes. These experiments also revealed a surprising capacity to label endogenous antigens in the absence of exogenous antigen stimulus or adjuvant in the AgRSR mouse. It prompted further investigations into how the TCR signal threshold controls Nur77 reporter activity. *in vitro* T cell stimulation assays demonstrate that the reporter activity not only had a broad dynamic range of signalling reporter activity but also a correlation with TCR signal strength *in vitro*; a finding corroborated using a dual reporter version of the AgRSR mouse generated specifically to label T cells in receipt of high and low TCR signals, both *in vitro* and *in vivo*. Collectively, these results highlight the AgRSR mouse's capacity to label T cells reactive to diverse antigens of a broad avidity range, previously elusive to traditional methods. As such, it represents an advancement in our technological armamentarium and has the potential to contribute to understanding a variety of disease settings. Amongst disease, cancer has been characterized as having T cell responses driven by unknown and weak antigenic signals. Exploiting the unique advantages of a system that both assesses T cell immunity across a broad dynamic range of antigenic strength and T cell immunity to unknown antigens, I investigated the tumour immune response. I applied the AgRSR system to a model of melanoma (YUMMER1.7) to understand how clonal dysfunction is achieved for CD8+ T cells in tumours. It was first demonstrated that, unlike bystander T cells marked before tumour implantation, tumour-relevant T cells marked after tumour implantation showed significant intratumoral expansion. Remarkably, an enrichment of EYFP+ T cells that are PD1+ in the spleen was also observed. This led us to hypothesize the presence of tumour-reactive T cells in the spleen and lymph node tissue that may be clonally related. Kinetic assessment of the EYFP+ cells later revealed a similar kinetic trend between the tissue sites, suggesting a similar dynamics of expansion and contraction exist between both systems. With the hypothesis that the CD8+ T cells in both spatial compartments of the tumour and secondary lymphoid tissues may contain identical clonotypes, I set out to develop workflows that link the selectivity of the AgRSR mouse to in-depth analysis of their transcriptome and clonality using single cell RNA sequencing (scRNAseq) to enable analysis of T cells at a clonal level. First, I generated robust protocols for the isolation and processing of EYFP+ cells to scRNAseq pipeline to overcome the challenge of preserving rare cells under time constraints. With these protocols established, I moved on to characterize these cells using combined scRNAseq and VDJseq by sampling the T cells at day 8 and day 18 after tamoxifen administration, at their respective peak and plateau of expansion. Bioinformatic analysis revealed the presence of T cell clones confined exclusively to the tumour and T cell clones distributed between the tumour and secondary lymphoid organs. These clones differed in their overall transcriptional state: with exhausted members compartmentalized in the tumour and functional effectors remaining in the secondary organs. Further comparison of the dynamics between day 8 and day 18 samples revealed a loss in the extent of clones distributed across the tumour and secondary lymphoid organs, suggesting that the supply of rejuvenating functional lymphoid tissues are lost over time. Finally, to establish directionality in the migratory relationship between the spatially confined effector T cells in the secondary lymphoid tissue and exhausted T cells in the tumour, I developed robust methods for intratumoral delivery of 4-hydroxytamoxifen to specifically label intratumoral CD8+ T cells and the isolation of those cells for scRNAseq analysis. This revealed an intriguing property of exhausted CD8+ T cells, that they do not egress from the tumour, in contrast to Treg cells that are freely detected in both tissues. This adds a spatial characteristic to the definition of T cell exhaustion. Taken together, this thesis established the utility of the AgRSR mouse in characterizing CD8+ T cell behaviours in tumours, permitting us to identify clonotype-independent invariant properties of T cell dysfunction. From the immunotherapeutic perspective, it can be concluded that a focus on rejuvenating the functional clones in the secondary lymphoid tissues may overcome current limitations in immunotherapies and cancer treatment. More broadly, the use of AgRSR mouse as a tool can pave the way for further interrogation of clonal principles towards other disease settings, fundamental principles regulating clone-dependent, and T-cell intrinsic behaviours can be distinguished.Item Open Access Investigations into the role of RNA modification in the function of the c-Myc IRESGoodacre, AlexanderDuring patho-physiological conditions, which lead to cell stress, there is a rapid and large reduction in protein synthesis rates. This is mediated, in part, by a decrease in cap-dependent initiation. In several cases, it has been shown that cap independent pathways are used for the translation of mRNAs required as part of the cell stress response/permit cell survival, and internal ribosome entry segments (IRES) have been shown to recruit the mRNAs to ribosomes directly in these conditions. It is known that the majority of cellular IRESs, unlike viral IRES, require a “nuclear event” before they are active the cytoplasm and recent data have shown that m6A methylation alone, which occurs in the nucleus, is sufficient to promote internal ribosome entry. Therefore, a testable hypothesised is that the “nuclear” event required for IRES function is RNA modification. Prior data from a yeast 3 Hybrid screen identified a novel S-adenosyl-L-methionine dependent methyltransferase Fibrillarin-Like-1 (FBLL1) as binding to the IRES of c-Myc. This protein, largely unstudied, shares homology with Fibrillarin; a well characterised 2’O-methyltransferase ubiquitously expressed and located in the nucleolus, consistent with its role in ribosomal RNA modification and, ribosomal biogenesis. Fibrillarin adds a methyl group to the 2' position of the ribose sugar of an RNA molecule. This modification can affect RNA stability, function and interactions with proteins and small molecules. Such interactions can either stabilize or destabilize the RNA. The c-Myc IRES contains a highly structured RNA sequence which is essential for its role in cap-independent translation. Therefore, this novel 2’O-methylatransferase, FBLL1, may be essential for regulating its complex structure. This thesis investigates the role of RNA modification, by FBLL1 and additional methyltransferases, on the activation of the c-Myc IRES. Recombinant FBLL1 was produced to investigate the direct effect of FBLL1 on the c-Myc IRES in vitro. In addition, proteins that "read" and "erase" such RNA modifications were also assessed for their impact on the expression of c-Myc protein, cell growth and global protein synthesis rates. Bicistronic reporter vectors were also used to specifically observe the effects of these altered methylation effects on a range of IRESs function including c-Myc, Bag1, MNT, Apaf-1, HCV and EMCV.Item Embargo Integrative multiomic insights into Alzheimer’s disease pathologyYu, Yizhou; Yu, Yizhou [0000-0002-4800-9392]Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder, currently afflicting around 900,000 patients in the United Kingdom. However, therapeutic avenues and factors that can exacerbate AD pathologies remain unclear. Hence, there is an urgent need for a better understanding of AD at the molecular, organellar, cellular, and behavioural levels. Here, I integrated insights from a fruit fly model of AD with data from AD patients. The accumulation of misfolded amyloid-β (Aβ) peptides as plaques is one of the main histopathological hallmarks of AD. In Chapter 3, I analysed the metabolomic changes in flies expressing human Aβ and uncovered alterations in nicotinamide adenine dinucleotide (NAD+) metabolism. NAD+, a component of vitamin B, is essential for both mitochondrial bioenergetics and DNA repair through NAD+-consuming poly (ADP-ribose) polymerases (PARPs). I found that increasing the bioavailability of NAD+ pharmacologically or by suppressing PARP is neuroprotective in fly models. Using UK Biobank data, I showed that polymorphisms in the human *PARP1* gene or the intake of vitamin B, are associated with a decrease in the risk and severity of Alzheimer’s disease. Building on these observations, I measured PAR levels in flies and found evidence of higher PARP activity. In Chapter 4, I hypothesised that higher PARP is linked to DNA misrepair, and validated this hypothesis in single-nuclei genome sequencing data from AD patients. I observed an imbalance in the nucleotide pool and showed that pharmacological or genetic enhancement of nucleotide metabolism is neuroprotective in AD. In Chapter 5, I conducted a proteomics analysis and revealed significant alterations in one-carbon (1C) metabolism. The 1C pathway facilitates the transfer of methyl groups for biosynthetic pathways and mitochondrial metabolism. I found that enhancing one-carbon metabolism pharmacologically or genetically decreases AD-related impairments in a fly model of AD, and corroborated these results using human data. During my PhD, I also observed and quantified large changes in sleep patterns in fly models, which recapitulate clinical phenotypes in AD patients. Upon comparing proteomics, metabolomics and single-cell RNA data in Chapter 6, I identified the redox-sensitive protein Hyperkinetic and its substrate NADP+ as potentially having a causative role in sleep changes in AD. I showed that overexpressing this gene and manipulating redox in the brain modulates sleep duration and AD pathology. Since my work highlighted the importance of mitochondrial function in AD, I tested whether drugs that cause mitochondrial dysfunction could have any toxic effects. In Chapter 7, I showed that the antipsychotic aripiprazole, which is taken by AD patients to treat psychotic symptoms, is neurotoxic through inhibiting mitochondrial complex I. Taken together, my work integrates multiomic data from animal models and human AD patients to gain insights into disease mechanisms, drug safety and therapeutic mechanisms. These results offer translational opportunities and call for further clinical validations to develop effective interventions for AD.Item Open Access Establishing new approaches to unveil regulatory functions of tRNAs and their interactorsMonti, MieThe discovery of the genetic code transformed the life sciences by providing an analytical framework to study living systems. However, once the role of tRNAs was established in protein synthesis, the field moved on to other areas of the RNA world. With the mainstream application of systems-wide approaches, tRNAs have emerged as implicated in multiple regulatory networks beyond translation. These roles have been associated to the variability in tRNA sequence and base modification. Here, a novel tRNA-seq. method was devised to accurately quantify the tRNAome and outline the modification and truncation landscapes, allowing to query the concerted response of tRNAs across conditions. The method was then applied to characterise the tRNA expression for an inherited mitocondropathy caused by incorrect tRNA processing in patients. Furthermore, recent studies have indicated that tRNA involvement beyond translation is also mediated by moonlighting functions of aminoacyl tRNA synthetases (aaRSs). For example, Tyrosyl-tRNA synthetase (YARS) was seen to translocate to the nucleus upon genotoxic stress. By applying a novel method (infSILAC), it is possible to quantitatively identify direct interactors and provide a solution to the problem of infinite ratios within a given SILAC dataset. The abundance of YARS-binding partners provided further evidence to the notion that aaRSs have functionally co-opted their tRNA binding site over evolutionary time. Next, whether the tRNAome also underpins essential biological processes within prokaryotes was explored. By assaying the RNA-binding proteome across the *E. coli* lifecycle stages it was possible to identify dynamic interactors. tRNA-modifying enzymes appeared responsive across conditions, including YfiF, a predicted methyl-transferase that was subsequently validated as RNA-binding *in vitro*. Molecular chaperones such as DnaK and HtpG, reported to bind tRNA molecules in human cell lines, also appeared in this study. By producing the first dynamic RBPome map in bacteria, hundreds of novel candidates have been identified that reconfigure their RNA-protein interactions between growth stages. Examining tRNA biology through multiple omics approaches and across domains, we lay the foundations for a more comprehensive understanding of the role of the tRNA molecules within cellular homeostasis and adaptation.Item Open Access 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.