Studies on the role of cholesterol in the pathogenesis of prion diseases: I. Correlation between cell susceptibility to prion infection/replication and altered cellular cholesterol distribution

The aim of the research project was to investigate whether cell susceptibility to prion infection/replication was somehow related to alterations in the intracellular cholesterol homeostasis, and in particular whether unbalanced FC vs. CE pools could be found in prion infected/prion susceptible cells with respect to cell conditions of no infection or genetic resistance

In vitro synergistic anti-prion effect of cholesterol ester modulators

Background. 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 compared to animals with a scrapie-resistant genotype, and that drugs influencing cholesterol esterification were endowed with selective anti-prion activity in N2a cell lines infected with the 22L and RML prion strains. Results. In 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. Comparative lipid analyses in prion-infected and non-infected N2a cells by colorimetric, enzymatic, and chemical means, clearly demonstrated a derangement of type and distribution of cholesterol esters, free cholesterol, and triglycerides in the infected N2a cells. Although single-drug treatments influenced lipid syntheses, only the combined-drug treatments appeared to restore a lipid profile similar to that of untreated-uninfected cells. Conclusions. We conclude that the anti-prion synergistic effect of cholesterol ester modulators with the cholesterol metabolism interfering anti-prion drugs chlorpromazine and quinacrine may arise from the ability of combined drugs to re-establish the intracellular lipid profile of untreated-uninfected 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

Altered cholesterol ester cycle in ex vivo skin fibroblasts from Alzheimer patients

Recent studies in both animal and cell models of Alzheimer's disease (AD) indicated that sub-cellular cholesterol distribution seems to regulate amyloid-beta (A[beta]) generation in the brain. In particular, cholesterol-esters (CE), rather than total cholesterol levels, appear directly correlated with A[beta] production. Here we observed that, similarly to brain cells, skin fibroblasts obtained from AD patients produce and accumulate more CE than skin fibroblasts from age-matched healthy controls do. AD fibroblasts also exhibited a 2 fold increase in the expression of ACAT1, in addition to lower levels of SREBP2, nCEH, Caveolin-1 and ABCA1 mRNA levels, all of which are involved in the CE cycle. HMGCoA-reductase and LDL-receptor mRNAs levels did not show statistically significant changes in AD, compared to non-AD, cells. Furthermore, although APP mRNA did not significantly vary, neprilysin (NEP), the most important enzyme in the proteolysis of A[beta], was expressed at very low levels in skin fibroblasts of sporadic AD patients. Our results contribute to the concept that AD may be the consequence of a basic and systemic defect in the CE cycle. Moreover, our results identify new possible targets for the diagnosis, prevention, and cure or, at least, amelioration of the symptoms of AD

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

ACAT-1, Cav-1 and PrP expression in scrapie susceptible and resistant sheep

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

Cholesterol Metabolism in Brain and Skin Fibroblasts from Sarda Breed Sheep With Scrapie-resistant and Scrapie-susceptible Genotypes

Scrapie is a fatal spongiform encephalopathy of sheep, a transmissible form of prion disease caused by neuronal accumulation of the aberrantly conformed prion protein (PrPsc). Currently, no ante-mortem diagnostic tests are available to detect this untreatable disease in the pre-clinical stage, thus making difficult to control its spread. Recent evidence suggests that the production of PrPsc can be modulated by the levels of membrane cholesterol in neuronal cells. Since cholesterol levels in cell membranes are dependent on cholesterol homeostasis in the whole organism, we studied cholesterol metabolism in brain tissues, plasma and skin fibroblasts of Sarda breed sheep with scrapie-resistant (ARR/ARR) and scrapie-susceptible (ARQ/ARQ) prion protein genotypes, both not infected (ARQ/ARQ-) and infected (ARQ/ARQ+) with scrapie. We found that, the levels of cytoplasmic cholesterol esters (CE) in brains and skin fibroblasts from sheep with the ARQ/ARQ genotype were consistently higher than those from sheep with the ARR/ARR genotype. Conversely, the levels of free cholesterol (FC) were lower in ARQ/ARQ, as compared to ARR/ARR sheep, thus resulting in a sharp reduction of the FC/CE ratio. Moreover, both uninfected and infected ARQ/ARQ sheep showed abnormally low levels of high density lipoprotein-cholesterol (HDL-C) in their plasma, as compared to ARR/ARR sheep. These data other than adding new strength to the notion that altered levels of intracellular cholesterol may indicate the presence of a lipid metabolic state that predisposes to infection with, and accumulation of, PrPsc in the brain, discriminate for the first time between two distinct but related cellular pools of cholesterol, namely membrane FC on one hand and cytoplasmic CE on the other. © 2007 Science Publications