[show abstract][hide abstract] ABSTRACT: Prion diseases are rare but invariably fatal neurodegenerative disorders. They are associated with spongiform encephalopathy, a histopathology characterized by the presence of large, membrane-bound vacuolar structures in the neuropil of the brain. While the primary cause is recognized as conversion of the normal form of prion protein (PrP(C)) to a conformationally distinct, pathogenic form (PrP(Sc)), the cellular pathways and mechanisms that lead to spongiform change, neuronal dysfunction and death are not known. Mice lacking the Mahogunin Ring Finger 1 (MGRN1) E3 ubiquitin ligase develop spongiform encephalopathy by 9 months of age but do not become ill. In cell culture, PrP aberrantly present in the cytosol was reported to interact with and sequester MGRN1. This caused endo-lysosomal trafficking defects similar to those observed when Mgrn1 expression is knocked down, implicating disrupted MGRN1-dependent trafficking in the pathogenesis of prion disease. As these defects were rescued by over-expression of MGRN1, we investigated whether reduced or elevated Mgrn1 expression influences the onset, progression or pathology of disease in mice inoculated with PrP(Sc). No differences were observed, indicating that disruption of MGRN1-dependent pathways does not play a significant role in the pathogenesis of transmissible spongiform encephalopathy.
PLoS ONE 01/2013; 8(1):e55575. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Brain aggregates (BrnAggs) derived from fetal mouse brains contain mature neurons and glial cells. We determined that BrnAggs are consistently infected with Rocky Mountain Laboratory scrapie strain prions and produce increasing levels of the pathogenic form of the prion protein (PrP). Their abundant dendrites undergo degeneration shortly after prion infection. Treatment of prion-infected BrnAggs with drugs, such as a γ-secretase inhibitors and quinacrine (Qa), which stop PrP formation and dendritic degeneration, mirrors the results from rodent studies. Because PrP is trafficked into lysosomes by endocytosis and autophagosomes by phagocytosis in neurons of prion strain-infected BrnAggs, we studied the effects of drugs that modulate subcellular trafficking. Rapamycin (Rap), which activates autophagy, markedly increased light-chain 3-II (LC3-II)-positive autophagosomes and cathepsin D-positive lysosomes in BrnAggs but could not eliminate the intracellular PrP within them. Adding Qa to Rap markedly reduced the number of LC3-II-positive autolysosomes. Rap + Qa created a competition between Rap increasing and Qa decreasing LC3-II. Rapamycin + Qa decreased total PrP by 56% compared with that of Qa alone, which reduced PrP by 37% relative to Rap alone. We conclude that the decrease was dominated by the ability of Qa to decrease the formation of PrP. Therefore, BrnAggs provide an efficient in vitro tool for screening drug therapies and studying the complex biology of prions.
Journal of Neuropathology and Experimental Neurology 04/2012; 71(5):449-66. · 4.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: The lipophilic cationic compound quinacrine has been used as an antimalarial drug for over 75 years but its pharmacokinetic profile is limited. Here, we report on the pharmacokinetic properties of quinacrine in mice. Following an oral dose of 40 mg/kg/day for 30 days, quinacrine concentration in the brain of wild-type mice was maintained at a concentration of ∼1 µM. As a substrate of the P-glycoprotein (P-gp) efflux transporter, quinacrine is actively exported from the brain, preventing its accumulation to levels that may show efficacy in some disease models. In the brains of P-gp-deficient Mdr1(0/0) mice, we found quinacrine reached concentrations of ∼80 µM without any signs of acute toxicity. Additionally, we examined the distribution and metabolism of quinacrine in the wild-type and Mdr1(0/0) brains. In wild-type mice, the co-administration of cyclosporin A, a known P-gp inhibitor, resulted in a 6-fold increase in the accumulation of quinacrine in the brain. Our findings argue that the inhibition of the P-gp efflux transporter should improve the poor pharmacokinetic properties of quinacrine in the CNS.
PLoS ONE 01/2012; 7(7):e39112. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prion-infected cells accumulate a heterogeneous population of aberrantly folded PrP conformers, including the disease-causing isoform (PrP(Sc)). We found that specific chemicals can modulate the levels of various PrP conformers in cultured cells. Positively charged polyamidoamines (dendrimers) eliminated protease-resistant (r) PrP(Sc) from prion-infected cells and induced the formation of insoluble, protease-sensitive PrP aggregates (designated PrP(A)). Larger, positively charged polyamidoamines more efficaciously induced the formation of PrP(A) and cleared rPrP(Sc), whereas negatively charged polyamidoamines neither induced PrP(A) nor cleared rPrP(Sc). Although the biochemical properties of PrP(A) were shown to be similar to protease-sensitive (s) PrP(Sc), bioassays of PrP(A) indicated that it is not infectious. Our studies argue that PrP(A) represents an aggregated PrP species that is off-pathway relative to the formation of rPrP(Sc). It remains to be established whether the formation of PrP(A) inhibits the formation of rPrP(Sc) by sequestering PrP(C) in the form of benign, insoluble aggregates.
Journal of Biological Chemistry 12/2009; 285(14):10415-23. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Quinacrine is a potent antiprion compound in cell culture models of prion disease but has failed to show efficacy in animal bioassays and human clinical trials. Previous studies demonstrated that quinacrine inefficiently penetrates the blood-brain barrier (BBB), which could contribute to its lack of efficacy in vivo. As quinacrine is known to be a substrate for P-glycoprotein multi-drug resistance (MDR) transporters, we circumvented its poor BBB permeability by utilizing MDR(0/0) mice that are deficient in mdr1a and mdr1b genes. Mice treated with 40 mg/kg/day of quinacrine accumulated up to 100 microM of quinacrine in their brains without acute toxicity. PrP(Sc) levels in the brains of prion-inoculated MDR(0/0) mice diminished upon the initiation of quinacrine treatment. However, this reduction was transient and PrP(Sc) levels recovered despite the continuous administration of quinacrine. Treatment with quinacrine did not prolong the survival times of prion-inoculated, wild-type or MDR(0/0) mice compared to untreated mice. A similar phenomenon was observed in cultured differentiated prion-infected neuroblastoma cells: PrP(Sc) levels initially decreased after quinacrine treatment then rapidly recovered after 3 d of continuous treatment. Biochemical characterization of PrP(Sc) that persisted in the brains of quinacrine-treated mice had a lower conformational stability and different immunoaffinities compared to that found in the brains of untreated controls. These physical properties were not maintained upon passage in MDR(0/0) mice. From these data, we propose that quinacrine eliminates a specific subset of PrP(Sc) conformers, resulting in the survival of drug-resistant prion conformations. Transient accumulation of this drug-resistant prion population provides a possible explanation for the lack of in vivo efficacy of quinacrine and other antiprion drugs.