Anti-prion activity found in beetle grub hemolymph of Trypoxylus dichotomus septentrionalis

No remedies for prion disease have been established, and the conversion of normal to abnormal prion protein, a key event in prion disease, is still unclear. Here we found that substances in beetle grub hemolymph, after they were browned by aging for a month or heating for hours, reduced abnormal prion protein (PrP) levels in RML prion-infected cells. Active anti-prion components in the hemolymph were resistant to protease treatment and had molecular weights larger than 100 kDa. Aminoguanidine treatment of the hemolymph abolished its anti-prion activity, suggesting that Maillard reaction products are enrolled in the activity against the RML prion. However, levels of abnormal PrP in RML prion-infected cells were not decreased by incubation with the Maillard reaction products formed by amino acids or bovine serum albumin. The anti-prion components in the hemolymph modified neither cellular or cell-surface PrP levels nor lipid raft or autophagosome levels. The anti-prion activity was not observed in cells infected with 22 L prion or Fukuoka-1 prion, suggesting the anti-prion action is prion strain-dependent. Although the active components of the hemolymph need to be further evaluated, the present findings imply that certain specific chemical structures in the hemolymph, but not chemical structures common to all Maillard reaction products, are involved in RML prion formation or turnover, without modifying normal PrP expression. The anti-prion components in the hemolymph are a new tool for elucidating strain-dependent prion biology.     

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.     

Promising developments bringing prion diseases closer to therapy and prophylaxis

Prion diseases are fatal, infectious, neurodegenerative disorders, and there are no available therapeutic or prophylactic regimens. The potential of immune system components in combating peripheral prion infection has long been underestimated, but recent studies have suggested that such molecules could be effective. For example, promising results have been reported from a passive vaccination study in prion-infected mice. In addition, elegant transgenic mouse studies have shown the inhibitory effect on prion propagation of a soluble immunoglobulin G (IgG)-like dimeric prion protein. This type of molecule might represent a new class of anti-prion compounds.     

Poly-L-histidine inhibits prion propagation in a prion-infected cell line

ABSTRACT

Transmissible spongiform encephalopathies (TSEs) are a group of lethal neurodegenerative diseases involving the structural conversion of cellular prion protein (PrP^C) into the pathogenic isoform (PrP^Sc) for which no effective treatment is currently available. Previous studies have implicated that a polymeric molecule with a repeating unit, such as pentosane polysulfate and polyamidoamide dendrimers, exhibits a potent anti-prion activity, suggesting that poly-(amino acid)s could be a candidate molecule for inhibiting prion propagation. Here, by screening a series of poly-(amino acid)s in a prion-infected neuroblastoma cell line (GT^FK), we identified poly-L-His as a novel anti-prion compound with an IC₅₀ value of 1.8 µg/mL (0.18 µM). This potent anti-prion activity was specific to a high-molecular-weight poly-L-His and absent in monomeric histidine or low-molecular-weight poly-L-His. Solution NMR data indicated that poly-L-His directly binds to the loop region connecting Helix 2 and Helix 3 of PrP^C and sterically blocks the structural conversion toward PrP^Sc. Poly-L-His, however, did not inhibit prion propagation in a prion-infected mouse when administered intraperitoneally, suggesting that the penetration of blood-brain barrier and/or the chemical stability of this polypeptide must be addressed before its application in vivo. Taken together, this study revealed the potential use of poly-L-His as a novel treatment against TSEs. (203 words)     

The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells

The conversion of the cellular prion protein (PrP(c)) into pathologic PrP(Sc) and the accumulation of aggregated PrP(Sc) are hallmarks of prion diseases. A variety of experimental approaches to interfere with prion conversion have been reported. Our interest was whether interference with intracellular signaling events has an impact on this conversion process. We screened approximately 50 prototype inhibitors of specific signaling pathways in prion-infected cells for their capacity to affect prion conversion. The tyrosine kinase inhibitor STI571 was highly effective against PrP(Sc) propagation, with an IC(50) of < or =1 microM. STI571 cleared prion-infected cells in a time- and dose-dependent manner from PrP(Sc) without influencing biogenesis, localization, or biochemical features of PrP(c). Interestingly, this compound did not interfere with the de novo formation of PrP(Sc) but activated the lysosomal degradation of pre-existing PrP(Sc), lowering the half-life of PrP(Sc) from > or =24 h to <9 h. Our data indicate that among the kinases known to be inhibited by STI571, c-Abl is likely responsible for the observed anti-prion effect. Taken together, we demonstrate that treatment with STI571 strongly activates the lysosomal degradation of PrP(Sc) and that substances specifically interfering with cellular signaling pathways might represent a novel class of anti-prion compounds.     

Antibodies to a Nonconjugated Prion Protein Peptide 95-123 Interfere with PrP<Superscript><Emphasis Type="Bold">Sc</Emphasis></Superscript> Propagation in Prion-Infected Cells

Summary 1. Vaccination-induced anti-prion protein antibodies are presently regarded as a promising approach toward treatment of prion diseases. Here, we investigated the ability of five peptides corresponding to three different regions of the bovine prion protein (PrP) to elicit antibodies interfering with PrP^Sc propagation in prion-infected cells. 2. Rabbits were immunized with free nonconjugated peptides. Obtained immune sera were tested in enzyme-linked immunosorbent assay (ELISA) and immunoblot for their binding to recombinant PrP and cell-derived pathogenic isoform (PrP^Sc) and normal prion protein (PrP^c), respectively. Sera positive in all tests were chosen for PrP^Sc inhibition studies in cell culture. 3. All peptides induced anti-peptide antibodies, most of them reacting with recombinant PrP. Moreover, addition of the serum specific to peptide 95–123 led to a transient reduction of PrP^Sc levels in persistently prion-infected cells. 4. Thus, anti-PrP antibodies interfering with PrP^Sc propagation were induced with a prion protein peptide nonconjugated to a protein carrier. These results point to the potential application of the nonconjugated peptide 95–123 for the treatment of prion diseases.     

Prion infection influences murine endogenous retrovirus expression in neuronal cells

Prions as causative agents of transmissible spongiform encephalopathies have been well investigated in experimental and modelling work. However, little is known about the molecular pathogenesis of prion-induced encephalopathies, the role of co-factors, and the interaction of prions with cellular components. We investigated the influence of prion infection on expression of murine endogenous retroviruses (ERVs), which compose approximately 10% of the mouse genome. Hypothalamic neuronal cells (GT1) and neuroblastoma cells (N2a) were examined. Both cell lines can be persistently infected with mouse adapted prion strains, i.e., RML. Using a mammalian retrovirus-specific DNA microarray and quantitative PCR methods, we compared the expression profiles of ERVs in prion-infected, uninfected, and anti-prion compound-treated murine neuronal cell lines, including clonal cell populations. The results suggest that prion infection influences ERV expression in neuronal cell lines, that this influence is cell line-specific, ERV-specific, and responsive to anti-prion compound treatment.     

Lithium induces clearance of protease resistant prion protein in prion-infected cells by induction of autophagy

Lithium is used for several decades to treat manic-depressive illness (bipolar affective disorder). Recently, it was found that lithium induces autophagy, thereby promoting the clearance of mutant huntingtin and α-synucleins in experimental systems. We show here for the first time that lithium significantly reduces the amount of pathological prion protein (PrP^Sc) in prion-infected neuronal and non-neuronal cultured cells by inducing autophagy. Treatment of prion-infected cells with 3-methyladenine, a potent inhibitor of autophagy, counteracted the anti-prion effect of lithium, demonstrating that induction of autophagy mediates degradation of PrP^Sc. Co-treatment with lithium and rapamycin, a drug widely used to induce autophagy, had an additive effect on PrP^Sc clearance compared to treatment with either drug alone. In addition, we provide evidence that the ability to reduce PrP^Sc and to induce autophagy is common for diverse lithium compounds, not only for the drug lithium chloride, usually administered in clinical therapy. Furthermore, we show here that besides reduction of PrP^Sc-aggregates, lithium-induced autophagy also slightly reduces the levels of cellular prion protein. Limiting the substrate available for conversion of cellular prion protein into PrP^Sc may provide an additional mechanism for reduction of PrP^Sc by lithium-induced autophagy.     

Intracellular re-routing of prion protein prevents propagation of PrP(Sc) and delays onset of prion disease

Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We show that the chemical compound Suramin induced aggregation of PrP in a post-ER/Golgi compartment and prevented further trafficking of PrP(c) to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re-routed to acidic compartments for intracellular degradation. In contrast to PrP(Sc) in prion-infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin-induced intracellular re-routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti-prion compounds.     

Disease-related prion protein forms aggresomes in neuronal cells leading to caspase activation and apoptosis

The molecular basis for neuronal death in prion disease is not established, but putative pathogenic roles for both disease-related prion protein (PrP(Sc)) and accumulated cytosolic PrP(C) have been proposed. Here we report that only prion-infected neuronal cells become apoptotic after mild inhibition of the proteasome, and this is strictly dependent upon sustained propagation of PrP(Sc). Whereas cells overexpressing PrP(C) developed cytosolic PrP(C) aggregates, this did not cause cell death. In contrast, only in prion-infected cells, mild proteasome impairment resulted in the formation of large cytosolic perinuclear aggresomes that contained PrP(Sc), heat shock chaperone 70, ubiquitin, proteasome subunits, and vimentin. Similar structures were found in the brains of prion-infected mice. PrP(Sc) aggresome formation was directly associated with activation of caspase 3 and 8, resulting in apoptosis. These data suggest that neuronal propagation of prions invokes a neurotoxic mechanism involving intracellular formation of PrP(Sc) aggresomes. This, in turn, triggers caspase-dependent apoptosis and further implicates proteasome dysfunction in the pathogenesis of prion diseases.     

KDEL-tagged anti-prion intrabodies impair PrP lysosomal degradation and inhibit scrapie infectivity

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPC) into the infectious scrapie isoform (PrPSc). We have recently demonstrated that anti-prion intrabodies targeted to the lumen of the endoplasmic reticulum provide a simple and effective means to inhibit the transport of PrPC to the cell surface. Here, we report that they completely block the traffic of mature full-length PrPC molecules, impair prion lysosomal degradation, and interfere with the early phase of scrapie formation. Since anti-prion intrabodies efficiently block PrPSc accumulation in vitro, we investigated whether they could also antagonize scrapie infectivity in vivo. We found that mice intracerebrally injected with KDEL-8H4-NGF-differentiated PC12 cells infected with scrapie neither develop scrapie clinical signs nor brain damage. Furthermore, no protease-resistant PrPSc is detectable in brains of inoculated animals. These results indicate that anti-prion intrabody strategy may be effective against prion infection.     

Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment

Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP^Sc). PrP^Sc is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP^C) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP^Sc (PrP^Sc-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP^Sc (PrP^Sc-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP^Sc can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119–127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP^Sc-sen and PrP^Sc-res even if all PrP^Sc molecules were not detected. The analytical dynamic range for PrP^Sc detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%–11% and 2.5–3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP^Sc detection did not affect the following PrP^Sc detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP^Sc detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.     

Prion Pathogenesis Is Faithfully Reproduced in Cerebellar Organotypic Slice Cultures

Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS) experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.     

Effective gene therapy in a mouse model of prion diseases

Classical drug therapies against prion diseases have encountered serious difficulties. It has become urgent to develop radically different therapeutic strategies. Previously, we showed that VSV-G pseudotyped FIV derived vectors carrying dominant negative mutants of the PrP gene are efficient to inhibit prion replication in chronically prion-infected cells. Besides, they can transduce neurons and cells of the lymphoreticular system, highlighting their potential use in gene therapy approaches. Here, we used lentiviral gene transfer to deliver PrPQ167R virions possessing anti-prion properties to analyse their efficiency in vivo. Since treatment for prion diseases is initiated belatedly in human patients, we focused on the development of a curative therapeutic protocol targeting the late stage of the disease, either at 35 or 105 days post-infection (d.p.i.) with prions. We observed a prolongation in the lifespan of the treated mice that prompted us to develop a system of cannula implantation into the brain of prion-infected mice. Chronic injections of PrPQ167R virions were done at 80 and 95 d.p.i. After only two injections, survival of the treated mice was extended by 30 days (20%), accompanied by substantial improvement in behaviour. This delay was correlated with: (i) a strong reduction of spongiosis in the ipsilateral side of the brain by comparison with the contralateral side; and (ii) a remarkable decrease in astrocytic gliosis in the whole brain. These results suggest that chronic injections of dominant negative lentiviral vectors into the brain, may be a promising approach for a curative treatment of prion diseases.     

Disease-associated prion protein oligomers inhibit the 26S proteasome

The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic beta subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the beta subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative beta sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein.     

Specific biarsenical labeling of cell surface proteins allows fluorescent- and biotin-tagging of amyloid precursor protein and prion proteins

Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins by using prion protein (PrP) and amyloid precursor protein (APP) as models. In prion-infected cells, FlAsH-labeled tetracysteine-tagged PrP converted from the normal isoform (PrPsen) to the disease-associated isoform (PrPres), suggesting minimal steric effects of the tag. Pulse-chase analysis of PrP and APP by fluorescent gel imaging demonstrated the utility of IDEAL labeling in investigating protein metabolism by identifying an as-yet-unrecognized C-terminal fragment (C3) of PrPsen and by characterizing the kinetics of PrPres and APP metabolism. C3 generation and N-terminal truncation of PrPres were inhibited by the anti-prion compound E64, a cysteine protease inhibitor. Surprisingly, E64 did not inhibit the synthesis of new PrPres, providing insight into the mechanism by which E64 reduces steady-state PrPres levels in prion-infected cells. To expand the versatility of tetracysteine tagging, we created new Alexa Fluor- and biotin-conjugated tetracysteine-binding molecules that were applied to imaging PrP endocytosis and ultrastructural localization. IDEAL-labeling extends the use of biarsenical derivatives to extracellular proteins and beyond microscopic imaging.