http://precedings.nature.com/users/ba8377580302ea1e1e699627e89a7645/ Documents posted by Alessandra Pani Nature Publishing Group en Nature Precedings http://precedings.nature.com/images/header_logo.gif http://precedings.nature.com http://precedings.nature.com/documents/3883/version/1 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. http://precedings.nature.com/documents/3883/version/1 Wed, 21 Oct 2009 15:17:23 UTC In vitro synergistic anti-prion effect of cholesterol ester modulators hdl:10101/npre.2009.3883.1 2009-10-21 Fabrizio Angius Nature Precedings 2009-10-21T15:17:23Z Manuscript Microbiology Neuroscience Pharmacology http://creativecommons.org/licenses/by/3.0/ http://precedings.nature.com/documents/1602/version/1 Recent studies in both animal and cell models of Alzheimer’s disease (AD) indicated that sub-cellular cholesterol distribution seems to regulate amyloid-beta (Aβ) generation in the brain. In particular, cholesterol-esters (CE), rather than total cholesterol levels, appear directly correlated with Aβ 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 Aβ, 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. http://precedings.nature.com/documents/1602/version/1 Tue, 19 Feb 2008 12:08:52 UTC Altered cholesterol ester cycle in ex vivo skin fibroblasts from Alzheimer patients hdl:10101/npre.2008.1602.1 2008-02-19 Alessandra Pani Nature Precedings 2008-02-19T12:08:52Z Manuscript Molecular Cell Biology Neuroscience http://creativecommons.org/licenses/by/3.0/ http://precedings.nature.com/documents/1246/version/1 Background. Previous epidemiological and experimental studies indicated cholesterol as a central player in Alzheimer disease (AD). Here, we utilized skin fibroblasts and PBMC as possible ex vivo models for the study of dysfunctions of cholesterol homeostasis which may be related to AD development. Methods. We analyzed cholesterol homeostasis using colorimetric, thin layer chromatography (TLC), and histologic technique in ex vivo cultures of skin fibroblasts and PBMCs from patients with probable AD and their first-degree relatives. Additionally, healthy age-matched individuals served as controls. Findings. As compared to controls, skin fibroblasts and PBMCs from AD patients, displayed an evident alteration of cholesterol metabolism; namely an anomalous accumulation of cholesterol esters in their cytoplasm. No change in intracellular free cholesterol was observed. Cellular overloading of cholesterol esters was dramatically increased after specific growth stimulation of the different cell types. Cholesterol ester accumulation was negatively correlated to plasma levels of high density lipoprotein cholesterol (HDL-C) and positively correlated with severity of cognitive symptoms measured by Mini-Mental State Examination (MMSE). Inhibitors of cholesterol esterification, such as progesterone and SaH, as well as a potent inhibitor of cell proliferation, RAD, were able to prevent accumulation of cholesterol esters. Interpretation. Changes of cholesterol esters in the peripheral compartment may be indicative of a systemic alteration of intracellular cholesterol homeostasis, which in turn might create a cellular milieu favourable to the production of ß-amyloid in the brain. Pathways that control cholesterol esterification might represent promising targets for novel diagnostic and therapeutic AD approaches. http://precedings.nature.com/documents/1246/version/1 Thu, 08 Nov 2007 10:41:33 UTC Changes in Cholesterol Metabolism in Peripheral Cells of Alzheimer Disease Patients and Their Relatives hdl:10101/npre.2007.1246.1 2007-11-08 Alessandra Pani Nature Precedings 2007-11-08T10:41:33Z Manuscript Molecular Cell Biology Neuroscience http://creativecommons.org/licenses/by/2.5/ http://precedings.nature.com/documents/1211/version/1 Scrapie is an infective ovine neurodegenerative disease; the only identified component of the infectious agent being an aberrant isoform (PrPSc) of the cellular prion protein (PrPC). So far, no means for ante-mortem diagnosis are available for Scrapie as well as for any other mammal Transmissible Spongiform Encephalopaties. We recently found a strong relationship between cell susceptibility to scrapie-infection and intracellular cholesterol homeostasis alterations. In brain tissues as well as in ex vivo cultures of skin fibroblasts and PBMCs from healthy and scrapie-affected sheep carrying a scrapie-susceptible (ARQ/ARQ) genotype, the levels of cholesterol esters were consistently higher than in tissues and cultures derived from animals with a scrapie-resistant (ARR/ARR) genotype. Moreover, both uninfected and scrapie-affected ARQ/ARQ sheep showed abnormally low levels of high density lipoprotein-cholesterol (HDL-C) in their plasma, as compared to ARR/ARR animals. We now show that intracellular accumulation of cholesterol esters in fibroblasts derived from scrapie-susceptible sheep was accompanied by parallel alterations in the expression level of genes and gene products (ACAT1 and Cav-1) that are involved in the pathways leading to intracellular cholesterol esterification and trafficking. Comparative analysis of PrPc mRNA, showed an higher expression level in cells from animals carrying susceptible genotype, with or without Scrapie. Preliminary experiments also revealed the presence of PK-resistant PrP isoforms in the latter cultures. The data reported in the present paper suggest that accumulation of cholesterol esters 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. http://precedings.nature.com/documents/1211/version/1 Tue, 09 Oct 2007 09:33:23 UTC ACAT1, Cav-1, and PrP expression in brains and skin fibroblasts from Sarda breed sheep with scrapie-resistant and scrapie-susceptible genotype. hdl:10101/npre.2007.1211.1 2007-10-09 Alessandra Pani Nature Precedings 2007-10-09T09:33:23Z Manuscript Molecular Cell Biology http://creativecommons.org/licenses/by/2.5/