A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein

Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrPC) into PrPSc, a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrPSc is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrPC may provide an opportunity to overcome these problems. PrPC ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrPC, and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrPC-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrPC-dependent synaptotoxicity of amyloid-beta (A beta) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrPC may produce a dual effect of blocking prion replication and inhibiting PrPC-mediated toxicity

Gerstmann-Sträussler-Scheinker disease subtypes efficiently transmit in bank voles as genuine prion diseases.

Gerstmann-Sträussler-Scheinker disease (GSS) is an inherited neurodegenerative disorder associated with mutations in the prion protein gene and accumulation of misfolded PrP with protease-resistant fragments (PrPres) of 6–8 kDa

Isolation of infectious, non-fibrillar and oligomeric prions from a genetic prion disease

Prions are transmissible agents causing lethal neurodegenerative diseases that are composed of aggregates of misfolded cellular prion protein (PrPSc). Despite non-fibrillar oligomers having been proposed as the most infectious prion particles, prions purified from diseased brains usually consist of large and fibrillar PrPSc aggregates, whose protease-resistant core (PrPres) encompasses the whole C-terminus of PrP. In contrast, PrPSc from Gerstmann-Sträussler-Scheinker disease associated with alanine to valine substitution at position 117 (GSS-A117V) is characterized by a small protease-resistant core, which is devoid of the C-terminus. We thus aimed to investigate the role of this unusual PrPSc in terms of infectivity, strain characteristics, and structural features. We found, by titration in bank voles, that the infectivity of GSS-A117V is extremely high (109.3 ID50 U/g) and is resistant to treatment with proteinase K (109.0 ID50 U/g). We then purified the proteinase K-resistant GSS-A117V prions and determined the amount of infectivity and PrPres in the different fractions, alongside the morphological characteristics of purified PrPres aggregates by electron microscopy. Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa PrPres, although the yield of PrPres was low. We found that this low yield depended on the low density/small size of GSS-A117V PrPres, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa PrPres, with infectivity levels that directly correlated with the PrPres amount detected. Finally, electron microscopy analysis of these fractions showed no presence of amyloid fibrils, but only very small and indistinct, non-fibrillar PrPresparticles were detected and confirmed to contain PrP via immunogold labelling. Our study demonstrates that purified aggregates of 7 kDa PrPres, spanning residues ∼90–150, are highly infectious oligomers that encode the biochemical and biological strain features of the original sample. Overall, the autocatalytic behaviour of the prion oligomers reveals their role in the propagation of neurodegeneration in patients with Gerstmann-Sträussler-Scheinker disease and implies that the C-terminus of PrPSc is dispensable for infectivity and strain features for this prion strain, uncovering the central PrP domain as the minimal molecular component able to encode infectious prions. These findings are consistent with the hypothesis that non-fibrillar prion particles are highly efficient propagators of disease and provide new molecular and morphological constraints on the structure of infectious prions

Pelodera strongyloides in the critically endangered Apennine brown bear (Ursus arctos marsicanus)

Skin biopsies from 20 Apennine brown bears (Ursus arctos marsicanus), 17 of which displaying skin lesions, were investigated by histopathology. Different degrees of dermatitis characterized by folliculitis and furunculosis accompanied by epidermal hyperplasia and epidermal and follicular hyperkeratosis were detected. In the most severe lesions, probably self induced by scratching, the superimposition of traumatic lesions was observed. In 8 out of 17 bears (47.0%) of affected bears, cross- and longitudinally-sectioned nematode larvae within the lumen of hair follicles were present, whose localization and morphological characteristics were consistent with Pelodera strongyloides. P. strongyloides is a free-living saprophytic nematode whose third-stage larvae can invade the skin causing pruritic dermatitis in several mammalian species. This is the first report of Pelodera infection in the brown bear. Although capable of causing primary dermatitis, the finding of Pelodera is not sufficient to conclude that it is the cause of the lesions observed in bears. Nevertheless, the high prevalence of the infection is indicative of a diffuse phenomenon that requires further specific investigations given the interest and conservational relevance of this relict bear population

Isolation of a Defective Prion Mutant from Natural Scrapie

<div><p>It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur <i>in vitro</i> during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the <i>in vitro</i> propagation of natural scrapie isolates. Although <i>in vitro</i> vole-adapted PrP^Sc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrP^Sc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrP^Sc conformers showed to be efficiently autocatalytic <i>in vitro</i> and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different <i>bona fide</i> PrP^Sc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for <i>in vivo</i> replication while the mutant was unable to infect voles, <i>de facto</i> behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least <i>in vitro</i>. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrP^C and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for <i>in vitro</i> replication. Finally, we propose a molecular mechanism able to account for the discordant <i>in vitro</i> and <i>in vivo</i> behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.</p></div

Identification of 14K from a natural scrapie sample.

<p><b>A</b>) Serial 10-fold dilutions of 2 Italian scrapie samples (ES47/10/3 and 198/9) were used as seeds in serial PMCA reactions using vole brain homogenate substrate. Products from rounds 4°, 6° and 8° (indicated in roman numbers) were digested with PK and analyzed by Western blot with antibody SAF84. After 8 PMCA rounds both samples were positive up to dilution 10⁻⁷. An atypical PrP^Sc with smaller PrP^res (indicated by the asterisk) emerged after the sixth round only from the last detectable dilution of sample 198/9, and was propagated until the end of the experiment. <b>B</b>) Three <i>in vitro</i> selected prion populations (18K, 14K/1 and 14K/2, as indicated on the top of each blot) were serially propagated for 4 successive PMCA rounds (represented in roman numbers). After each round, aliquots of the PMCA products were digested with PK and analyzed by Western blot with antibody SAF84. For 14K/1, PK-digested PrP^res is also shown after enzymatic removal of N-linked oligosaccarides, which allows to better appreciate the co-presence and evolution of 14K and 18K PrP^res (indicated by a single and a double asterisk respectively) during the experiment.</p

Vole bioassay.

<p><b>A</b>, <b>B</b> and <b>C</b> show the neuropathological and PrP^res phenotypes observed in voles after primary transmission (left panels, and indicated as I, roman number, in the blot on the right) and second passage (central panels and indicated as II, roman numbers, in the blot) of sheep 198/9 (<b>A</b>), PMCA-derived 18K (<b>B</b>) and PMCA-derived 14K/1 (<b>C</b>). Brain-scoring areas in lesion profiles are: medulla (1), cerebellum (2), superior colliculus (3), hypothalamus (4), thalamus (5), hippocampus (6), septum (7), retrosplenial and adjacent motor cortex (8), cingulate and adjacent motor cortex (9). For each blot a vole adapted scrapie was added (BvScr, last lane). PrP^res was detected by antibody SAF84. <b>D</b>) Graph depicting the denaturation profiles obtained by CSA from PrP^Sc in sheep 198/9 (Sh198/9) or from voles infected with 18K (Bv18K) and vole-adapted 198/9 (Bv198/9). <b>E</b>) Graph depicting the comparison of denaturation profiles of 18K before (18K) and after (Bv18K) transmission in voles. [GdnHCl]_1/2 values are reported in the graph. <b>F</b>) Graph depicting the fate of 18K and 14K/2 after intracerebral inoculation in voles. Two groups of 8 voles were inoculated with 14K/2 or 18K and 2 voles for each group were sacrificed at different time points (0, 3, 14 and 52 dpi). Their brains were homogenized and used as seed for PMCA reactions in 3 independent experiments. The values on y axis represent the overall percentage of positive samples per time point after 3 PMCA rounds.</p

Isolation and biochemical characterization of 18K and 14K PrP^Sc.

<p><b>A</b>) Serial ten-fold dilutions of 18K and 14K/1 PrP^res (10⁻² to 10⁻⁷) were subjected to 4 successive PMCA rounds (expressed in roman numbers) and PrP^res was analyzed by Western blot after each round. While 18K PrP^res alone was detectable in the whole set of dilutions seeded with 18K (upper side of the panel), both the PrP^res types emerged from sample 14K/1, i.e. 18K PrP^res from the low dilutions of the curve and 14K PrP^res from the high ones, indicating the mixed nature of the sample. <b>B</b>) Serial ten-fold dilutions of sample 14K/2 were propagated for 3 PMCA rounds. PrP^res obtained after the 2^nd and 3^rd rounds faithfully preserved the 14K signature. <b>C</b>) In order to compare their <i>in vitro</i> autocatalytic efficiency, serial ten-fold dilutions of 18K and 14K/2 were subjected to a single PMCA round, PK digested, analyzed by Western blot and the PrP^res detected with antibody SAF84. For each curve the inoculum not subjected to PMCA was added (first lane, F). <b>D</b>) Epitope mapping of PrP^res from vole-adapted scrapie (BvScr), 18K and 14K/2. PrP^C from a negative vole brain homogenate not treated with PK was also included as control. Membranes probed with Sha31 and SAF84 antibodies are shown, as indicated on each blot. <b>E</b>) Representative Western blot of CSA experiments with 18K and 14K/2. Aliquots of the samples were treated with increasing concentrations of GdnHCl (from 0 to 4 M) and residual PrP^res was analyzed by Western blot. <b>F</b>) Dose-response curves were obtained by plotting the mean fraction of PrP^res detected as a function of GdnHCl concentration and best fitted using a four parameter logistic equation. [GdnHCl]_1/2 values are indicated in the graph.</p

Hypothetical mechanisms underpinning the defective nature of 14K PrP^Sc.

<p>The cartoon depicts the hypothetical interaction between physiological proteases responsible for α-cleavage, here supposed to belong to the family of ADAM proteases [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006016#ppat.1006016.ref035" target="_blank">35</a>] and 18K or 14K PrP^Sc (<b>A</b>), and how it changes in the <i>in vitro</i> (<b>B</b>) and the <i>in vivo</i> (<b>C</b>) replication environments. Distinct symbols indicate PrP^C monomers, 18K or 14K PrP^Sc aggregates, as well as the α-cleavage site and the location of the polybasic domains in PrP^Sc, as shown in the graphical legend below the cartoon. (<b>A</b>) In 18K PrP^Sc aggregates the physiological α-cleavage site (residues 110–111, vole PrP numbering) and the central polybasic domain (in green, aa ~ 101–110), are tightly packed in the PK-resistant core of PrP^Sc. On the contrary, in mutant 14K PrP^Sc the α-cleavage site is available for hydrolysis. (<b>B</b>) During <i>in vitro</i> propagation by PMCA, the activity of ADAM proteases is purposely prevented by protease inhibitors, a factor which allows to keep full length PrP in solution. Under these conditions, both 18K and 14K PrP^Sc are full length and preserve intact polybasic domains, which would allow them to interact with PrP^C and replicate. (<b>C</b>) <i>In vivo</i>, 18K PrP^Sc is still protected from ADAM proteolysis as the cleavage site is buried within the PK-resistant core, and thus it is still fully competent for replication as it retains the N-terminus. In contrast, 14K PrP^Sc would be cleaved at 110–111 and would lose the central polybasic domain, supposed to be a key mediator of the interaction with partners indispensable for prion replication (PrP^C or cofactors).</p

Transmission of 198/9, 18K, 14K/1 and 14K/2 in bank voles.

<p>Transmission of 198/9, 18K, 14K/1 and 14K/2 in bank voles.</p