Medicine, Dentistry & Health Sciences Collected Works - Theses
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ItemHIV and prions: two distinct infections targeting host cholesterol metabolism( 2014)It is well established that pathogens target host cholesterol metabolism in order to survive and replicate. The perturbation of host cellular cholesterol metabolism caused by pathogens can lead to metabolic complications, such as atherosclerosis and neurodegenerative diseases. The aim of this study was to elucidate the mechanisms responsible for perturbation of intracellular cholesterol metabolism caused by pathogens. The mechanistic understanding of this process is essential as it may provide new insight into pathogenesis of infections and their metabolic complications and new approaches for targeting cholesterol metabolism as a new treatment for both infection and their associated metabolic disorders. In this study we investigated the effects of two dissimilar pathogens, HIV and prion, on the cholesterol removal process, known as the Reverse Cholesterol Transport (RCT) pathway. It has been shown that both HIV and prion rely heavily on host cholesterol during the replication cycle. Although they are two very dissimilar pathogens, both utilise lipid rafts in the host cell in order to successfully replicate. It was hypothesised that both HIV and prion pathogens target the RCT pathway in order to acquire sufficient cholesterol to successfully replicate. In this thesis, it was shown that both HIV and the prion pathogen targets the ATP binding cassette transporter A1 (ABCA1) dependent cholesterol trafficking pathway; by diverting intracellular cholesterol to lipid raft domains, thereby stabilising the lipid raft platform for further pathogen propagation. It was shown that both HIV and prion infection inhibits ABCA1 dependent cholesterol efflux. The mechanism behind this functional impairment is the displacement of ABCA1 from the cell surface into the intracellular compartments, which significantly reduces the abundance of ABCA1 on the cell surface that is available to participate in cholesterol efflux. HIV infection significantly increased the risk of atherosclerosis by impairing the cholesterol efflux pathway. The viral protein responsible for the deregulation of cholesterol metabolism is Nef. RAW264.7 murine macrophages and RAW264.7 cells stably transfected with Nef were used to investigate the mechanism responsible for perturbation of cholesterol metabolism. Transfection of macrophages with Nef led to an enhanced movement of cellular cholesterol to lipid rafts and an increased abundance of rafts as validated by using three independent techniques. The knockdown of ABCA1 expression by siRNA or inhibition ABCA1 function by chemical compounds further promotes cholesterol trafficking towards lipid rafts, suggesting Nef does not use ABCA1 for transporting cholesterol to rafts, but rather competes with ABCA1 for cholesterol. Furthermore, it has been shown that Nef affects functionality of ABCA1 by mislocalisation of ABCA1. Nef caused the redistribution of ABCA1 between raft and non-raft fractions of the plasma membrane, and decreased the surface exposure of ABCA1. Furthermore, Nef increased colocalisation of ABCA1 with the lysosome marker LAMP-1, suggesting that Nef interacts with ABCA1 by redirecting it into lysosomes for degradation, resulting in an accumulation of cholesterol. Moreover, two mouse models of atherosclerosis (C57/BL6 and apoE-/-) were used to further investigate the effects of Nef on the development of atherosclerosis. Both in-vivo mouse models further support the notion that Nef significantly increased the risk of atherosclerosis by increasing cholesterol accumulation in macrophages, while having limited effects on inflammation at both systemic (in the plasma) and local (within atherosclerotic lesions) levels. Systemically, Nef also caused a sharp elevation of triglyceride level. Taken these results together, the combination of dyslipidemia and hypertriglyceridemia further support that Nef may also be a contributor to HIV induced atherosclerosis. During prion infection, conversion of prion protein (PrPC) into a pathological isoform (PrPSc) occurs in lipid rafts and is dependent on cholesterol. Here, it was shown that prion infection promotes an increased abundance of ABCA1. Despite this, cholesterol efflux in both prion infected mouse neuronal and fibroblast cells was reduced, consequently leading to an accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was also confirmed in prion-infected mice. Mechanistically, conversion of PrPC to the pathological isoform led to PrPSc accumulation in rafts and a displacement of ABCA1 from rafts and the cell surface toward intracellular compartments. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, where cholesterol plays an important role in the pathogenesis of the infection and its effect on neuronal cells. Given the importance of ABCA1 in the pathogenesis of HIV and prion infection, treatment with LXR agonist or overexpression of ABCA1 may significantly reduce the rate of HIV and/or prion infection. It appears that, in addition to maintaining cellular cholesterol content and restricting raft formation, the ABCA1 transporter also plays a role of an innate immune factor against HIV and prions. Given that many intracellular pathogens critically depend on rafts in their pathogenesis, increasing ABCA1 expression can be potentially used as an anti-infection therapeutic strategy.