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Liver injury instigates a proinflammatory response in tissue-resident macrophages, called Kupffer cells (KCs), resulting in the recruitment of monocytes and neutrophils. The high energy demand required for a rapid proinflammatory response in macrophages like KCs is achieved through metabolic reprogramming. This is supported by increased glycolysis. On the other hand, injury resolution requires hepatic macrophages to undergo an anti-inflammatory polarization, which relies on oxidative phosphorylation (OXPHOS). In addition to shifts in mechanisms of adenosine triphosphate (ATP) production, lipid metabolic reprogramming supplies metabolic intermediates and lipids for membrane remodeling and the production of inflammatory mediators. AMP-activated protein kinase (AMPK) is a master metabolic regulator that influences the metabolic reprogramming of macrophages. While AMPK activation promotes an anti-inflammatory polarization, disruption of activity exacerbates proinflammatory signaling. For this thesis work, we addressed whether macrophage AMPK is protective against liver injury by altering immunometabolism. Specifically, we investigated this question in the context of chronic (nonalcoholic steatohepatitis (NASH)) and acute (acetaminophen (APAP) overdose) liver injury. While APAP overdose is a robust and directly translational model of acute injury, models of NASH-induced hepatic fibrosis rely on nutrient-deficient diets like the choline-deficient high-fat diet (CDAHFD) or genetic manipulation. Despite the utility of these models, they seldom mirror the pathogenesis of human NASH, with diets like CDAHFD being completely dissociated from metabolic syndrome. Moreover, models are required to address the divergence between male and female mice. Recently, there has been a shift towards addressing other variables that drive inflammation and metabolism. At room temperature (RT) (22 °C), mice experience cold stress that alters various biological functions. Cold stress drives brown adipose tissue (BAT) activation and upregulates corticosterone production and immunosuppression, all processes that blunt NASH progression. Giles et al. (2016) demonstrated that housing mice at thermoneutrality (TN) (30 °C) exacerbated metabolic-dysfunction associated fatty liver disease (MAFLD) progression toward NASH in both male and female mice. Since then, we and others have implemented TN housing with different dietary interventions and mice strains. We determined that 16-week Western diet (WD) feeding of male and female mice at 29 °C was insufficient to drive hepatic fibrosis, however alterations in glucose tolerance and elevated liver injury enzymes as well as profibrotic gene expression in male mice may indicate that a longer timeline is necessary (24 weeks). Given that our TN NASH model did not produce hepatic fibrosis, we implemented the CDAHFD to investigate macrophage AMPK in chronic liver injury. Male and female AMPK Flox (Prkaa1 fl/fl/Prkaa2 fl/fl) and MacKO (Flox-LysM-Cre+) mice were fed CDAHFD for 8 weeks. In this time frame, CDAHFD produces a lean euglycemic phenotype with hepatic steatosis, inflammation, and fibrosis, to which AMPK MacKO had no influence. Moreover, intervention with a low dose of metformin had no effect, contrary to the reduction in hepatic steatosis observed in HFD-fed mice. Although macrophage AMPK is dispensable in the CDAHFD model of chronic liver injury, acute liver injury needed to be addressed. We found that priming with systemic activation of a direct AMPK activator MK-8722 did not influence hepatic injury and necrosis in our model of APAP-induced liver injury (AILI). Moreover, deletion of hepatocellular AMPK (Flox-Alb-Cre+) or AMPK MacKO did not influence injury at 24 hours post overdose. Despite the lack of effect of systemic AMPK activation, we were interested in a nanoparticle-based targeting of direct AMPK activator MK-8722 (NP-MK8722) delivery. We determined that PLGA-PEG nanoparticles (NPs) accumulated in hepatic macrophages as early as 2 hours post-injection, but NP-MK8722 did not alter hepatic necrosis, injury, or immune infiltration. Overall, my thesis work has advanced our knowledge of the effects of housing temperatures on NASH pathogenesis. Moreover, we are the first to address the effects of macrophage AMPK signaling in NASH and AILI. This is especially true for assessing how AMPK deficiency and targeted activation influences KC immunometabolism during injury.

Homologous Recombination (HR) is a major double-stranded break (DSB) DNA repair pathway. It utilizes the sister chromatid as a template, thus promoting error-free repair. The initial HR step is DNA end resection with the creation of RPA coated single-stranded DNA (ssDNA) overhangs. This is followed by RAD51 loading onto the resected DNA, triggering strand invasion, and homology search. The BRCA1 protein is fundamental for HR mediated DNA repair, and patients with germline BRCA1 mutations have a high risk of developing cancer. These BRCA1 mutant tumors are HR deficient, and as a result, sensitive to PARP inhibitors (PARPi). BRCA1 likely promotes DNA end resection by displacing 53BP1 from the DSBs and also promotes RAD51 loading by enabling the formation of the larger BRCA1-PALB2-BRCA2 complex trough its coiled-coil region. Although BRCA1 functions in both HR steps, its essential role in each step is unclear, with previous reports showing that RAD51 loading after DNA end resection occurred could be BRCA1 independent. Here, we demonstrated that full HR restoration after activation of end resection mediated by 53BP1 knockout (KO), requires the expression of a BRCA1 protein that retained the coiled-coil region and its protein interactions. We observed that although 53bp1 KO can restore the Mendelian frequencies of a Brca1 null mouse model, mice were still tumor prone, PARPi sensitive, and had low levels of RAD51 γ-irradiation-induced foci (IRIF), suggesting that HR levels were minimal. Moreover, in order to significantly increase PARPi resistance in human cancer cell lines, 53BP1 loss of function (LoF) needs to be associated with the expression of a hypomorphic BRCA1 protein that retained the PALB2 interaction. We also demonstrated that Brca1 compound heterozygosity could rescue the developmental defects observed in Brca1 mutant homozygous mice, as long as the alleles retained complementary HR-related functions. Additionally, these compound heterozygotes mice were not tumor prone and had a normal lifespan, suggesting that HR was restored. In contrast, when the allele combination did not exhibit complementary functions, the developmental defects persisted. Overall, we demonstrated that to fully function in mouse development, tumor suppression, and drug resistance, BRCA1 role is critical in both the DNA end resection and the RAD51 loading HR steps. / Biomedical Sciences

Synucleins are highly conserved presynaptic proteins with unknown function. α-synuclein plays a key role regulating dopamine homeostasis and is intimately involved in Parkinson’s disease (PD) pathogenesis. However, the normal/pathological role of α-synuclein remains unidentified. Studies exploring its function are limited as current transgenic mouse models do not fully recapitulate PD pathology. This thesis reports the functional characterisation of two novel synuclein-based mouse models. I report the molecular and functional characterisation of transgenic mouse lines with wild-type or A30P-mutant human α-synuclein genomic locus carried within a bacterial artificial chromosome. SNCA-A30P^+Snca-/- mice exhibited a highly physiologically relevant expression pattern of the transgene, including expression in the substantia nigra pars compacta (SNpc) and a specific, age-related loss of TH^+ cells in the SNpc, the key region of preferential cell loss in PD, compared with non-transgenic Snca -/- littermate controls. Analysis of dopamine signalling using fast-scan cyclic voltammetry (FCV) showed young adult SNCA-A30P^+Snca-/- mice had an approximately 20% lower evoked extracellular dopamine concentration ([DA]o) compared with non-transgenic Snca -/- littermate controls, a decrease specific to the dorsal striatum. This difference diminished with age and could not be attributed to changes in dopamine reuptake/content. I detail the behavioural and neurochemical phenotype in mice lacking all three synucleins (α/β/γ). Functional compensation between synucleins emphasises the importance of studying their effects by removing all three proteins simultaneously. Triple-null mice exhibited hyperactivity in a novel environment reminiscent of a hyperdopaminergic-like phenotype, but showed no phenotype in anxiety or motor related tests. FCV revealed synuclein triple-null mice had a two-fold increase in [DA]o, specific to the dorsal striatum and not attributable to changes in dopamine reuptake/content, changes in striatal nicotinic receptor activity nor calcium-dependent changes in dopamine exocytosis. Together, the analysis from these two novel mouse models reveal synucleins play an important role in altering synaptic function in the dorsal striatum (the region selectively affected in PD) and contributes to growing evidence suggesting synucleins are negative regulators of synaptic dopamine release.

Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.