Search

Abstract Background Cholesterol homeostasis dysfunction has been reported to have role in the pathogenesis of Alzheimer disease (AD). Therefore, changes in cholesterol metabolism in blood components may help to develop new potential AD biomarkers. In this study changes in cholesterol metabolism-related gene expression genes were evaluated in peripheral blood mononuclear cells (PBMCs) from AD subjects, their first degree relatives (FDR) and two groups of age matched controls (C1 > 80 years, C2 < 60 years). The expression of three genes related to APP processing was also determined. Results Results showed significantly different behavior (P = 0.000) in the expression of all analyzed genes among the 4 groups. An inverse correlation emerged between the age of controls and the propensity of their PBMCs to express selected genes. Moreover, when gene expression was evaluated in PBMCs from AD patients and compared with that of PBMCs from healthy subjects of the same age, LDL-R and APP mRNAs were most abundant in AD as compared C1 whereas SREBP-2 and particularly nCEH were present at much lower mRNA levels in AD-PBMCs. This study describes for the first time a differential expression profile of cholesterol and APP related genes in PBMCs from AD patients and their FDR. Conclusions We suggest that the expressions of cholesterol homeostasis and APP processing related genes in PBMC could be proposed as possible biomarkers to evaluate AD risk. In addition, gene expression in PBMC could be also used for diagnosis and development of therapeutic strategies as well as for personalized prediction in clinical outcome of AD.

Abstract Objective Cholesterol changes have been described in prion-cell models and in experimental rodent scrapie; yet, the pattern of this association is still controversial. Methods To shed light on the matter, we analysed and compared cholesterol variations in ScN2a cells and in brains of Scrapie-infected C57Bl/6 mice, using two different methods: a fluorimetric-enzymatic cholesterol assay, and high performance liquid chromatography-mass spectroscopy (HPLC-MS). Results Compared to uninfected controls, similar cholesterol metabolism anomalies were observed in infected cells and brains by both methods; however, only HPLC-MS revealed statistically significant cholesterol variations, particularly in the cholesteryl esters (CE) fraction. HPLC-MS analyses also revealed different fatty acid composition of the CE fraction in cells and brains. In N2a cells, their profile reflected that of serum, while in normal brains cholesteryl-linoleate only was found at detectable levels. Following prion infection, most CE species were increased in the CE pool of ScN2a cells, whereas a conspicuous amount of cholesteryl-arachidonate only was found to contribute to the cerebral increase of CE. Of interest, oral pravastatin administration to Scrapie-infected mice, was associated with a significant reduction of cerebral free cholesterol (FC) along with a concomitant further increase of the CE pool, which included increased amounts of both cholesteryl-linoleate and cholesteryl-arachidonate. Conclusion Although mechanistic studies are needed to establish the pathophysiological relevance of changes in cerebral CE concentrations, to the best of our knowledge this is the first report to provide evidence of increased cholesterol esterification in brains of prion-infected mice, untreated and treated with pravastatin.

Abstract Background Alzheimer's disease is the most common progressive neurodegenerative disease. In recent years, numerous progresses in the discovery of novel Alzheimer's disease molecular biomarkers in brain as well as in biological fluids have been made. Among them, those involving lipid metabolism are emerging as potential candidates. In particular, an accumulation of neutral lipids was recently found by us in skin fibroblasts from Alzheimer's disease patients. Therefore, with the aim to assess whether peripheral alterations in cholesterol homeostasis might be relevant in Alzheimer's disease development and progression, in the present study we analyzed lipid metabolism in plasma and peripheral blood mononuclear cells from Alzheimer's disease patients and from their first-degree relatives. Methods Blood samples were obtained from 93 patients with probable Alzheimer's disease and from 91 of their first-degree relatives. As controls we utilized 57, cognitively normal, over-65 year-old volunteers and 113 blood donors aged 21-66 years, respectively. Data are reported as mean ± standard error. Statistical calculations were performed using the statistical analysis software Origin 8.0 version. Data analysis was done using the Student t-test and the Pearson test. Results Data reported here show high neutral lipid levels and increased ACAT-1 protein in about 85% of peripheral blood mononuclear cells freshly isolated (ex vivo) from patients with probable sporadic Alzheimer's disease compared to about 7% of cognitively normal age-matched controls. A significant reduction in high density lipoprotein-cholesterol levels in plasma from Alzheimer's disease blood samples was also observed. Additionally, correlation analyses reveal a negative correlation between high density lipoprotein-cholesterol and cognitive capacity, as determined by Mini Mental State Examination, as well as between high density lipoprotein-cholesterol and neutral lipid accumulation. We observed great variability in the neutral lipid-peripheral blood mononuclear cells data and in plasma lipid analysis of the subjects enrolled as Alzheimer's disease-first-degree relatives. However, about 30% of them tend to display a peripheral metabolic cholesterol pattern similar to that exhibited by Alzheimer's disease patients. Conclusion We suggest that neutral lipid-peripheral blood mononuclear cells and plasma high density lipoprotein-cholesterol determinations might be of interest to outline a distinctive metabolic profile applying to both Alzheimer's disease patients and asymptomatic subjects at higher risk of disease.

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrPSc) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrPSc propagation involves conversion from its normal isoform, PrPC, by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrPSen) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrPSc (PrPRes). Scrapie brain dilutions up to 10¯8 and 10¯13 were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrPRes levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrPC. Although the brains of 263K-affected mice had little immunoblot-detectable PrPRes, RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrPRes strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrPRes levels. We also found that eQuIC, which incorporates a PrPSc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrPSc. [ABSTRACT FROM AUTHOR]

Our studies on the role of cholesterol in prion infection/replication showed that brains and peripheral cells of sheep susceptible-to or suffering-from Scrapie were characterized by an altered cholesterol homeostasis, and that drugs affecting cholesterol ester pool were endowed with selective anti-prion activity in N2a cell lines infected with the 22L and RML prion strains. In these prion-infected N2a cell lines, we now report increased anti-prion activity of dual-drug combinations consisting of cholesterol ester modulators associated with prion inhibitors. Synergism was obtained with the cholesterol ester modulators everolimus, pioglitazone, progesterone, and verapamil associated with the anti-prion chlorpromazine, and with everolimus and pioglitazone associated with the anti-prion quinacrine. In addition, comparative lipid analyses in prion-infected vs. uninfected N2a cells, demonstrated a derangement of type and distribution of cholesterol ester, free cholesterol, and triglyceride pools in the infected cells. Single-drug treatments differently affected synthesis of the various lipid forms, whereas combined drug treatments appeared to restore a lipid profile similar to that of the untreated-uninfected cells. We conclude that the anti-prion synergistic effects of cholesterol ester modulators associated with the cholesterol-interfering anti-prion drugs chlorpromazine and quinacrine may arise from the ability of combined drugs to re-establish lipid homeostasis in the prion-infected cells. Overall, these data suggest that inhibition of prion replication can be readily potentiated by combinatorial drug treatments and that steps of cholesterol/cholesterol ester metabolism may represent suitable targets. [ABSTRACT FROM AUTHOR]

Scrapie is a prion disease for which no means of ante-mortem diagnosis is available. We recently found a relationship between cell susceptibility to scrapie and altered cholesterol homeostasis. In brains and in skin fibroblasts and peripheral blood mononuclear cells from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype, the levels of cholesterol esters were consistently higher than in tissues and cultures derived from animals with a scrapie-resistant genotype. Here we show that intracellular accumulation of cholesterol esters (CE) in fibroblasts derived from scrapie-susceptible sheep was accompanied by parallel alterations in the expression level of acyl-coenzymeA: cholesterol-acyltransferase (ACAT1) and caveolin-1 (Cav-1) that are involved in the pathways leading to intracellular cholesterol esterification and trafficking. Comparative analysis of cellular prion protein (PrPc) mRNA, showed an higher expression level in cells from animals carrying a susceptible genotype, with or without Scrapie. These data suggest that CE accumulation in peripheral cells, together with the altered expression of some proteins implicated in intracellular cholesterol homeostasis, might serve to identify a distinctive lipid metabolic profile associated with increased susceptibility to develop prion disease following infection. [ABSTRACT FROM AUTHOR]