Sarah Vascellari

New generation QuIC assays for prion seeding activity

The ability of abnormal TSE-associated forms of PrP to seed the formation of amyloid fibrils from recombinant PrPSen has served as the basis for several relatively rapid and highly sensitive tests for prion diseases. These tests include rPrP-PMCA (rPMCA), standard quaking-induced conversion (S-QuIC), amyloid seeding assay (ASA), real-time QuIC (RT-QuIC) and enhanced QuIC (eQuIC). Here, we summarize recent improvements in the RT-QuIC-based assays that enhance the practicality, sensitivity and quantitative attributes of assays QuIC and promote the detection of prion seeding activity in dilute, inhibitor-laden fluids such as blood plasma.

Prion Seeding Activities of Mouse Scrapie Strains with Divergent PrPSc Protease Sensitivities and Amyloid Plaque Content Using RT-QuIC and eQuIC

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.

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 threefold-higher 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 [EC50] range, 1.4 to 40 μM) were comparable to those of antiprion reference compounds (EC50 range, 0.6 to 10 μM). 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.

Synthesis, structural characterization, formation constants and in vitro cytotoxicity of phenanthroline and imidazolidine-2-thione copper(II) complexes

The synthesis, crystal structures, physicochemical properties and complex formation constants of [Cu(phen)(2)(L)](ClO(4))(2) complexes, where phen is 1,10-ortho-phenanthroline and L is a series of substituted imidazolidine-2-thione, have been studied. Single crystal X-ray diffraction revealed a distorted trigonal-bipyramidal geometry for all the molecules. The complex formation constants were determined in nonaqueous media by spectrophotometric measurements. Testing copper(II) complexes in mouse neuroblastoma N2a cells persistently infected with the 22L strain of the scrapie prion protein (22L-N2a) resulted in high cytotoxicity but no antiprion activity at nontoxic doses.

Novel copper(II) complexes as new promising antitumour agents. A crystal structure of [Cu(1,10-phenanthroline-5,6-dione)2(OH2)(OClO3)](ClO4).

The cytotoxic properties of copper(II) complexes with 1,10-phenanthroline (phen) can be modified by substitution in the phen backbone. For this purpose, Cu(II) complexes with phen, 1,10-phenanthrolin-5,6-dione (phendione) and 1,10-phenanthrolin-5,6-diol (phendiol) have been synthesised and characterised. The crystal structure of [Cu(phendione)2(OH2)(OClO3)](ClO4) is discussed. The complex formation equilibria between Cu(II) and phen or phendione were studied by potentiometric measurements at 25 and 37°C in 0.1 M ionic strength (NaCl). The antitumour activity of the compounds has been tested in vitro against a panel of tumour (DU-145, HEP-G2, SK-MES-1, CCRF-CEM, CCRF-SB) and normal (CRL-7065) human cell lines. The studied compounds generally present an antiproliferative effect greater than that of cisplatin. The phen and phendione ligands present a similar antiproliferative effect against all the tested cells. Phendiol presents an antiproliferative effect 1.3 to 18 times greater than that of phen or phendione for leukemic, lung, prostatic and fibroblast cells, while it presents less activity towards hepatic cells. Complexes with two ligands are more cytotoxic towards all the tested cell lines than complexes with one ligand and are generally more cytotoxic than the ligand alone. Complexes [Cu(phendiol)2(OH2)](ClO4)2 and [Cu(phendione)2(OH2)(OClO3)](ClO4) appear to be the most active compounds for the treatment of SK-MES-1 and HEP-G2 cells, respectively, being at least 18 times more cytotoxic than cisplatin. The studied Cu(II) complexes are characterised by a strong DNA affinity and were found to interact with DNA mainly by groove binding or electrostatic interactions. The complexes appear to act on cells with a mechanism different from that of cisplatin.

Glucose-6-phosphate-dehydrogenase Deficiency as a Risk Factor for Pterygium

PURPOSE Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway (PPP), providing reducing power (NADPH) and pentose phosphates. The purpose of this study was to investigate the possible involvement of G6PD deficiency (G6PD-) in the pathogenesis of pterygium. METHODS Erythrocyte G6PD activity was evaluated in 123 pterygium patients and in 112 age-matched control patients. Enzyme activity, mRNA, rate of growth, green autofluorescence, response to oxidative stress, and cholesterol metabolism were determined in pterygium fibroblasts (PFs) and in normal conjunctival fibroblasts (NCFs) isolated from G6PD normal (NCFs+ and PFs+) and G6PD- (NCFs- and PFs-) patients. RESULTS Higher prevalence of G6PD- was found in patients affected by primary pterygium than in control subjects, both men and women, suggesting that this enzymatic defect may be a predisposing factor for pterygium. G6PD activity was significantly lower in NCFs- than in NCFs+, but not in PFs- than in PFs+. In PFs-, G6PD mRNA levels were significantly higher than in PFs+. Growth-stimulated NCFs- grew at half the rate of NCFs+, although PFs- and PFs+ grew at the same rate. Increased green autofluorescence and susceptibility to oxidative stress were observed in PFs (+/-) and in NCFs-, but not in NCFs+. Moreover, ex vivo PFs (+/-) accumulated more lipids than corresponding NCFs. CONCLUSIONS The results of this study, although restricted to a limited group of subjects (i.e., those of Sardinian ancestry), suggest that G6PD- not only does not protect against pterygium, but may even be considered a risk factor for the development of this disorder.

PrP knockout cells expressing transmembrane PrP resist prion infection

ABSTRACT Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrPC) influences PrPC misfolding into the disease-associated isoform, PrPres, as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrPC away from rafts. Previous studies showed that nonraft transmembrane PrPC variants resist conversion to PrPres when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrPC and GPI-anchored PrPres in distinct membrane environments. Thus, it remained unclear whether transmembrane PrPC might convert to PrPres if seeded by an exogenous source of PrPres not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrPC. To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrPC supported persistent PrPres propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrPC is required for persistent PrPres propagation, implicating raft microdomains as a location for conversion. IMPORTANCE Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrPC or transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPres. We show that only GPI-anchored PrPC was able to convert to PrPres and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrPC to rafts are crucial to the ability of PrPC to propagate as a prion.

Accumulation and aberrant composition of cholesteryl esters in Scrapie-infected N2a cells and C57BL/6 mouse brains

ObjectiveCholesterol changes have been described in prion-cell models and in experimental rodent scrapie; yet, the pattern of this association is still controversial.MethodsTo 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).ResultsCompared 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.ConclusionAlthough 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.

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

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.

Novel coumarins and related copper complexes with biological activity: DNA binding, molecular docking and in vitro antiproliferative activity

Coumarins show biological activity and are widely exploited for their therapeutic effects. Although a great number of coumarins substituted by heterocyclic moieties have been prepared, few studies have been carried out on coumarins containing pyridine heterocycle, which is known to modulate their physiological activities. We prepared and characterized three novel 3-(pyridin-2-yl)coumarins and their corresponding copper(II) complexes. We extended our investigations also to three known similar coumarins, since no data about their biochemical activity was previously been reported. The antiproliferative activity of the studied compounds was tested against human derived tumor cell lines and one human normal cell line. The DNA binding constants were determined and docking studies with DNA carried out. Selected Quantitative Structure-Activity Relationship (QSAR) descriptors were calculated in order to relate a set of structural and topological descriptors of the studied compounds to their DNA interaction and cytotoxic activity.