title: Investigating how metabolite fluxes affect two brain diseases using different model systems
Creators: name: Gao, Xuexin
Publisher: name: The Australian National University
Year: 2024

description: The work described in this thesis investigated the metabolite fluxes involved in two brain diseases, divided into two parts. Part A studied metabolite flux of ketone bodies in a glioblastoma cell line and compared with other cancer cell lines. Investigation of Part A concentrated at cellular level and the flux of ketone body metabolism was examined to illustrate how cells use ketone bodies for support of proliferation. The study was expected to tell us whether a ketogenic diet may be beneficial to glioblastoma treatment. Glioblastoma is an aggressive form of brain cancer, presenting significant challenges due to limited treatment options. While normal brain cells primarily utilise glucose for energy, they can adapt to alternative substrates such as ketone bodies. This study investigates whether different cancer cell lines retain the ability to utilise ketone bodies as energy substrates, with a focus on beta-hydroxybutyrate metabolism. The results revealed that all cell lines exhibited enhanced aerobic glycolysis, leading to lactate production in the presence of oxygen, with compensatory flux from glutamine metabolism into the TCA cycle. Supplementing beta-hydroxybutyrate did not promote the growth of any cell line, despite the expression of requisite transporters and enzymes. Beta-hydroxybutyrate contributed to metabolic flux into the TCA cycle for biomass and energy production; however, this pathway was significantly reduced in the glioblastoma cell line U-87 MG compared to the other cell lines. This finding suggests the potential efficacy of ketogenic diets as a treatment option for glioblastoma patients, providing additional energy substrates for normal cell metabolism. Furthermore, the U-87 MG cell line exhibited overexpression of the mitochondrial uncoupling protein UCP2 and distinct morphological changes in mitochondria, indicating a potential link between these factors and the reduced utilisation of beta-hydroxybutyrate through the TCA cycle. Further investigation is warranted to elucidate this relationship fully. Part B investigated the efflux of amyloid-beta peptides out of cells. Deposition of amyloid-beta peptides is one of the hallmarks of Alzheimer's disease and it may relate to an impaired clearance pathway with a critical actor: P-glycoprotein. This investigation focused on the organismal level and used C. elegans as a model organism for measuring behavioural effects. The expectation was to elucidate whether P-glycoprotein could be a target in the prevention of Alzheimer's disease. Alzheimer's disease poses a growing concern to society, particularly with the aging population. Current understanding implicates abnormal accumulation of amyloid-beta peptides as a key factor in its pathogenesis, potentially leading to neuronal dysfunction. Our observation indicated that the presence of amyloid-beta aggregates in the body wall muscles or somatic nervous system correlated with impaired locomotion, neuromuscular functions, and a shortened lifespan in C. elegans. A C. elegans strain with human P-glycoprotein expression in body wall muscles was made and used to generate a hybrid strain expressing both amyloid-beta and human P-glycoprotein. The expression of human P-glycoprotein in C. elegans caused paralysis and compromised neuromuclular functions in the worms, but the lifespan was unaffected. Although human P-glycoprotein expression did not reduce the accumulation of amyloid-beta peptides in worms, it restored locomotion compared to parental strains expressing amyloid-beta or human P-glycoprotein alone. The restoration of locomotion did not translate into improvements in neuromuscular function or lifespan. The results suggest that targeting the P-glycoprotein-mediated clearance pathway holds promise for preventing Alzheimer's disease. However, it also highlights the complexity of the clearance pathway, indicating that multiple components may be considered for effective therapeutic strategy.