Shortest known prion protein allele in highly BSE-susceptible lemurs

We describe the shortest prion protein allele known to date. Surprisingly, it is found as a polymorphism exactly in a species (prosimian lemurs) which seems highly susceptible to oral infection with BSE-derived prions. The truncation of the prion protein we found raises several questions. First, is the truncated octarepeat structure we describe, consisting of two octarepeats, still functional in copper binding? A second question is whether this truncation is related to the remarkable oral infectibility of lemurs with BSE-derived prions. And finally, one could argue that this genotype alone might favour development of a prion disease, even in the absence of exogenous infection

Excited-state dynamics of bacteriorhodopsin probed by broadband femtosecond fluorescence spectroscopy

AbstractThe impact of varying excitation densities (∼0.3 to ∼40 photons per molecule) on the ultrafast fluorescence dynamics of bacteriorhodopsin has been studied in a wide spectral range (630–900 nm). For low excitation densities, the fluorescence dynamics can be approximated biexponentially with time constants of <0.15 and ∼0.45 ps. The spectrum associated with the fastest time constant peaks at 650 nm, while the 0.45 ps component is most prominent at 750 nm. Superimposed on these kinetics is a shift of the fluorescence maximum with time (dynamic Stokes shift). Higher excitation densities alter the time constants and their amplitudes. These changes are assigned to multi-photon absorptions

Inhibition of cholesterol recycling impairs cellular PrPSc propagation

The infectious agent in prion diseases consists of an aberrantly folded isoform of the cellular prion protein (PrPc), termed PrPSc, which accumulates in brains of affected individuals. Studies on prion-infected cultured cells indicate that cellular cholesterol homeostasis influences PrPSc propagation. Here, we demonstrate that the cellular PrPSc content decreases upon accumulation of cholesterol in late endosomes, as induced by NPC-1 knock-down or treatment with U18666A. PrPc trafficking, lipid raft association, and membrane turnover are not significantly altered by such treatments. Cellular PrPSc formation is not impaired, suggesting that PrPSc degradation is increased by intracellular cholesterol accumulation. Interestingly, PrPSc propagation in U18666A-treated cells was partially restored by overexpression of rab 9, which causes redistribution of cholesterol and possibly of PrPSc to the trans-Golgi network. Surprisingly, rab 9 overexpression itself reduced cellular PrPSc content, indicating that PrPSc production is highly sensitive to alterations in dynamics of vesicle trafficking

Anti-prion drug mPPIg5 inhibits PrP(C) conversion to PrP(Sc).

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future

Docosahexaenoic and eicosapentaenoic acids increase prion formation in neuronal cells

<p>Abstract</p> <p>Background</p> <p>The transmissible spongiform encephalopathies, otherwise known as prion diseases, occur following the conversion of the cellular prion protein (PrP^C) to an alternatively folded, disease-associated isoform (PrP^Sc). Recent studies suggest that this conversion occurs via a cholesterol-sensitive process, as cholesterol synthesis inhibitors reduced the formation of PrP^Scand delayed the clinical phase of scrapie infection. Since polyunsaturated fatty acids also reduced cellular cholesterol levels we tested their effects on PrP^Scformation in three prion-infected neuronal cell lines (ScGT1, ScN2a and SMB cells).</p> <p>Results</p> <p>We report that treatment with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) or the cholesterol synthesis inhibitor simvastatin reduced the amounts of free cholesterol in membrane extracts from prion-infected neuronal cells. Simvastatin reduced cholesterol production while DHA and EPA promoted the conversion of free cholesterol to cholesterol esters. Crucially, while simvastatin reduced PrP^Scformation, both DHA and EPA significantly increased the amounts of PrP^Scin these cells. Unlike simvastatin, the effects of DHA and EPA on PrP^Sccontent were not reversed by stimulation of cholesterol synthesis with mevalonate. Treatment of ScGT1 cells with DHA and EPA also increased activation of cytoplasmic phospholipase A₂and prostaglandin E₂production. Finally, treatment of neuronal cells with DHA and EPA increased the amounts of PrP^Cexpressed at the cell surface and significantly increased the half-life of biotinylated PrP^C.</p> <p>Conclusion</p> <p>We report that although treatment with DHA or EPA significantly reduced the free cholesterol content of prion-infected cells they significantly increased PrP^Scformation in three neuronal cell lines. DHA or EPA treatment of infected cells increased activation of phospholipase A₂, a key enzyme in PrP^Scformation, and altered the trafficking of PrP^C. PrP^Cexpression at the cell surface, a putative site for the PrP^Scformation, was significantly increased, and the rate at which PrP^Cwas degraded was reduced. Cholesterol depletion is seen as a potential therapeutic strategy for prion diseases. However, these results indicate that a greater understanding of the precise relationship between membrane cholesterol distribution, PrP^Ctrafficking, cell activation and PrP^Scformation is required before cholesterol manipulation can be considered as a prion therapeutic.</p

Selective Processing and Metabolism of Disease-Causing Mutant Prion Proteins

Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrPC). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrPSc form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrPC to PrPSc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrPSc in familial prion disease

Self-avoiding walks and connective constants

The connective constant $\mu(G)$ of a quasi-transitive graph $G$ is the asymptotic growth rate of the number of self-avoiding walks (SAWs) on $G$ from a given starting vertex. We survey several aspects of the relationship between the connective constant and the underlying graph $G$. $\bullet$ We present upper and lower bounds for $\mu$ in terms of the vertex-degree and girth of a transitive graph. $\bullet$ We discuss the question of whether $\mu\ge\phi$ for transitive cubic graphs (where $\phi$ denotes the golden mean), and we introduce the Fisher transformation for SAWs (that is, the replacement of vertices by triangles). $\bullet$ We present strict inequalities for the connective constants $\mu(G)$ of transitive graphs $G$, as $G$ varies. $\bullet$ As a consequence of the last, the connective constant of a Cayley graph of a finitely generated group decreases strictly when a new relator is added, and increases strictly when a non-trivial group element is declared to be a further generator. $\bullet$ We describe so-called graph height functions within an account of "bridges" for quasi-transitive graphs, and indicate that the bridge constant equals the connective constant when the graph has a unimodular graph height function. $\bullet$ A partial answer is given to the question of the locality of connective constants, based around the existence of unimodular graph height functions. $\bullet$ Examples are presented of Cayley graphs of finitely presented groups that possess graph height functions (that are, in addition, harmonic and unimodular), and that do not. $\bullet$ The review closes with a brief account of the "speed" of SAW.Comment: Accepted version. arXiv admin note: substantial text overlap with arXiv:1304.721

Loss of Octarepeats in Two Processed Prion Pseudogenes in the Red Squirrel, Sciurus vulgaris

The N-terminal region of the mammalian prion protein (PrP) contains an ‘octapeptide’ repeat which is involved in copper binding. This eight- or nine-residue peptide is repeated four to seven times, depending on the species, and polymorphisms in repeat number do occur. Alleles with three repeats are very rare in humans and goats, and deduced PrP sequences with two repeats have only been reported in two lemur species and in the red squirrel, Sciurus vulgaris. We here describe that the red squirrel two-repeat PrP sequence actually represents a retroposed pseudogene, and that an additional and older processed pseudogene with three repeats also occurs in this species as well as in ground squirrels. We argue that repeat numbers may tend to contract rather than expand in prion retropseudogenes, and that functional prion genes with two repeats may not be viable

Antihypertensive Drug Guanabenz Is Active In Vivo against both Yeast and Mammalian Prions

Background: Prion-based diseases are incurable transmissible neurodegenerative disorders affecting animals and humans. [br/] Methodology/Principal Findings: Here we report the discovery of the in vivo antiprion activity of Guanabenz (GA), an agonist of a2-adrenergic receptors routinely used in human medicine as an antihypertensive drug. We isolated GA in a screen for drugs active in vivo against two different yeast prions using a previously described yeast-based two steps assay. GA was then shown to promote ovine PrPSc clearance in a cell-based assay. These effects are very specific as evidenced by the lack of activity of some GA analogues that we generated. GA antiprion activity does not involve its agonist activity on a2-adrenergic receptors as other chemically close anti-hypertensive agents possessing related mechanism of action were found inactive against prions. Finally, GA showed activity in a transgenic mouse-based in vivo assay for ovine prion propagation, prolonging slightly but significantly the survival of treated animals. [br/] Conclusion/Significance: GA thus adds to the short list of compounds active in vivo in animal models for the treatment of prion-based diseases. Because it has been administrated for many years to treat hypertension on a daily basis, without major side-effects, our results suggest that it could be evaluated in human as a potential treatment for prion-based diseases