Antiprion Activity of Cholesterol Esterification Modulators: a Comparative Study Using Ex Vivo Sheep Fibroblasts and Lymphocytes and Mouse Neuroblastoma Cell Lines

Abstract

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefoldhigher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC 50 ] range, 1.4 to 40 M) were comparable to those of antiprion reference compounds (EC 50 range, 0.6 to 10 M). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

In vitro synergistic anti-prion effect of cholesterol ester modulators in combination with chlorpromazine and quinacrine

“…These results confirm and extend our previous findings in the same cell model [14] and in the ovine scrapie [11][12][13], and further support the presence of a tight association between prion infection/susceptibility and altered cholesterol homeostasis. In agreement with our results, altered cholesterol metabolism/esterification with over-expression of ACAT-1 (soat1) was reported in mice brains during the course of experimental scrapie [34, refs therein], and up-regulation of cholesterol biosynthesis was recently described following prion A total of 30x10 6 of each cell culture was collected from sub-confluent flasks.…”

“…The effect on intracellular lipids of a drug combination with marked anti-PrP res synergism, such as everolimus (EVE, 0.02 µM) and chlorpromazine (CP, 2 µM), was evaluated by staining the cells with dyes with affinity for different lipids (Oil Red O for neutral lipids; Nile red for neutral lipids and phospholipids; Filipin for free cholesterol). As expected [14], Oil Red O (ORO) staining revealed significantly higher neutral lipids (NL) in the 22L-infected than in the uninfected N2a cells ( Figure 4A and B). However, both single and combined drug treatments markedly reduced red ORO intensity in the 22L-infected cell line.…”

“…In addition, in persistently prion-infected N2a cells, we found that drugs able to reduce the CE pool, i.e. everolimus, pioglitazone, Sandoz 58035, progesterone, and verapamil, were also able to inhibit PrP res formation [14]. We now report on the anti-prion effect of everolimus, pioglitazone, progesterone, and verapamil in dual-drug combinations with selected prion inhibitors.…”

“…CE and triglycerides, TG), but not free cholesterol (FC). ORO staining was performed in 60% isopropyl alcohol, followed by Mayer's hematoxylin counterstaining for nuclei [14]. After staining, cells were imaged using a Leitz inverted-phase microscope fitted with a digital camera.…”

Epitope-specific anti-prion antibodies upregulate apolipoprotein E and disrupt membrane cholesterol homeostasis

“…In more recent reports, we have shown that prion infection significantly increases levels of free cholesterol and of cPLA 2 in neuroblastoma cell lines (Bate et al, 2008c). Moreover, abnormal accumulation of cholesterol esters in peripheral cells has been observed in scrapie-infected sheep and treatment with compounds known to interfere with cholesterol homeostasis lead to significant reduction of cholesterol esters in these cells (Pani et al, 2007), indicating that cholesterol esterification is an important component in the protection against prion-mediated cell death (Bate et al, 2008a). In this present study, epitope-specific anti-prion antibodies (Beringue et al, 2003) have been utilized on cell culture models (Pleasure et al, 1992) on rat dorsal root ganglia neurons (DRG) and mouse primary cortical neurons (MPCN) in order to assess antibody-mediated effects on cholesterol homeostasis.…”