Florey Department of Neuroscience and Mental Health - Research Publications
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ItemDocosahexaenoic and eicosapentaenoic acids increase prion formation in neuronal cells(BIOMED CENTRAL LTD, 2008-09-12)BACKGROUND: The transmissible spongiform encephalopathies, otherwise known as prion diseases, occur following the conversion of the cellular prion protein (PrPC) to an alternatively folded, disease-associated isoform (PrPSc). Recent studies suggest that this conversion occurs via a cholesterol-sensitive process, as cholesterol synthesis inhibitors reduced the formation of PrPSc and delayed the clinical phase of scrapie infection. Since polyunsaturated fatty acids also reduced cellular cholesterol levels we tested their effects on PrPSc formation in three prion-infected neuronal cell lines (ScGT1, ScN2a and SMB cells). RESULTS: We report that treatment with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) or the cholesterol synthesis inhibitor simvastatin reduced the amounts of free cholesterol in membrane extracts from prion-infected neuronal cells. Simvastatin reduced cholesterol production while DHA and EPA promoted the conversion of free cholesterol to cholesterol esters. Crucially, while simvastatin reduced PrPSc formation, both DHA and EPA significantly increased the amounts of PrPSc in these cells. Unlike simvastatin, the effects of DHA and EPA on PrPSc content were not reversed by stimulation of cholesterol synthesis with mevalonate. Treatment of ScGT1 cells with DHA and EPA also increased activation of cytoplasmic phospholipase A2 and prostaglandin E2 production. Finally, treatment of neuronal cells with DHA and EPA increased the amounts of PrPC expressed at the cell surface and significantly increased the half-life of biotinylated PrPC. CONCLUSION: We report that although treatment with DHA or EPA significantly reduced the free cholesterol content of prion-infected cells they significantly increased PrPSc formation in three neuronal cell lines. DHA or EPA treatment of infected cells increased activation of phospholipase A2, a key enzyme in PrPSc formation, and altered the trafficking of PrPC. PrPC expression at the cell surface, a putative site for the PrPSc formation, was significantly increased, and the rate at which PrPC was degraded was reduced. Cholesterol depletion is seen as a potential therapeutic strategy for prion diseases. However, these results indicate that a greater understanding of the precise relationship between membrane cholesterol distribution, PrPC trafficking, cell activation and PrPSc formation is required before cholesterol manipulation can be considered as a prion therapeutic.
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ItemGlimepiride Reduces the Expression of PrPC, Prevents PrPSc Formation and Protects against Prion Mediated Neurotoxicity(PUBLIC LIBRARY SCIENCE, 2009-12-09)BACKGROUND: A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrP(C)) into a disease related, alternatively folded isoform (PrP(Sc)). The accumulation of PrP(Sc) within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases. PRINCIPAL FINDINGS: Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrP(C) from the surface of prion-infected neuronal cells. The cell surface is a site where PrP(C) molecules may be converted to PrP(Sc) and glimepiride treatment reduced PrP(Sc) formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82-146. Glimepiride treatment significantly reduce both the amount of PrP82-146 that bound to neurones and PrP82-146 induced activation of cytoplasmic phospholipase A(2) (cPLA(2)) and the production of prostaglandin E(2) that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrP(C) expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC. CONCLUSIONS: Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrP(Sc) formation and neuronal damage in prion diseases.
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ItemGinkgolides protect against amyloid-β1-42-mediated synapse damage in vitro(BIOMED CENTRAL LTD, 2008-01-07)BACKGROUND: The early stages of Alzheimer's disease (AD) are closely associated with the production of the Abeta1-42 peptide, loss of synapses and gradual cognitive decline. Since some epidemiological studies showed that EGb 761, an extract from the leaves of the Ginkgo biloba tree, had a beneficial effect on mild forms of AD, the effects of some of the major components of the EGb 761 extract (ginkgolides A and B, myricetin and quercetin) on synapse damage in response to Abeta1-42 were examined. RESULTS: The addition of Abeta1-42 to cortical or hippocampal neurons reduced the amounts of cell associated synaptophysin, a pre-synaptic membrane protein that is essential for neurotransmission, indicating synapse damage. The effects of Abeta1-42 on synapses were apparent at concentrations approximately 100 fold less than that required to kill neurons; the synaptophysin content of neuronal cultures was reduced by 50% by 50 nM Abeta1-42. Pre-treatment of cortical or hippocampal neuronal cultures with ginkgolides A or B, but not with myrecitin or quercetin, protected against Abeta1-42-induced loss of synaptophysin. This protective effect was achieved with nanomolar concentrations of ginkgolides. Previous studies indicated that the ginkgolides are platelet-activating factor (PAF) receptor antagonists and here we show that Abeta1-42-induced loss of synaptophysin from neuronal cultures was also reduced by pre-treatment with other PAF antagonists (Hexa-PAF and CV6209). PAF, but not lyso-PAF, mimicked the effects Abeta1-42 and caused a dose-dependent reduction in the synaptophysin content of neurons. This effect of PAF was greatly reduced by pre-treatment with ginkgolide B. In contrast, ginkgolide B did not affect the loss of synaptophysin in neurons incubated with prostaglandin E2. CONCLUSION: Pre-treatment with ginkgolides A or B protects neurons against Abeta1-42-induced synapse damage. These ginkgolides also reduced the effects of PAF, but not those of prostaglandin E2, on the synaptophysin content of neuronal cultures, results consistent with prior reports that ginkgolides act as PAF receptor antagonists. Such observations suggest that the ginkgolides are active components of Ginkgo biloba preparations and may protect against the synapse damage and the cognitive loss seen during the early stages of AD.
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ItemSequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A2 activation(BIOMED CENTRAL LTD, 2008-02-12)BACKGROUND: The transmissible spongiform encephalopathies (TSEs), otherwise known as the prion diseases, occur following the conversion of the normal cellular prion protein (PrPC) to an alternatively folded isoform (PrPSc). The accumulation of PrPSc within the brain leads to neurodegeneration through an unidentified mechanism. Since many neurodegenerative disorders including prion, Parkinson's and Alzheimer's diseases may be modified by cholesterol synthesis inhibitors, the effects of prion infection on the cholesterol balance within neuronal cells were examined. RESULTS: We report the novel observation that prion infection altered the membrane composition and significantly increased total cholesterol levels in two neuronal cell lines (ScGT1 and ScN2a cells). There was a significant correlation between the concentration of free cholesterol in ScGT1 cells and the amounts of PrPSc. This increase was entirely a result of increased amounts of free cholesterol, as prion infection reduced the amounts of cholesterol esters in cells. These effects were reproduced in primary cortical neurons by the addition of partially purified PrPSc, but not by PrPC. Crucially, the effects of prion infection were not a result of increased cholesterol synthesis. Stimulating cholesterol synthesis via the addition of mevalonate, or adding exogenous cholesterol, had the opposite effect to prion infection on the cholesterol balance. It did not affect the amounts of free cholesterol within neurons; rather, it significantly increased the amounts of cholesterol esters. Immunoprecipitation studies have shown that cytoplasmic phospholipase A2 (cPLA2) co-precipitated with PrPSc in ScGT1 cells. Furthermore, prion infection greatly increased both the phosphorylation of cPLA2 and prostaglandin E2 production. CONCLUSION: Prion infection, or the addition of PrPSc, increased the free cholesterol content of cells, a process that could not be replicated by the stimulation of cholesterol synthesis. The presence of PrPSc increased solubilisation of free cholesterol in cell membranes and affected their function. It increased activation of the PLA2 pathway, previously implicated in PrPSc formation and in PrPSc-mediated neurotoxicity. These observations suggest that the neuropathogenesis of prion diseases results from PrPSc altering cholesterol-sensitive processes. Furthermore, they raise the possibility that disturbances in membrane cholesterol are major triggering events in neurodegenerative diseases.