Isolation of Proteinase K-Sensitive Prions Using Pronase E and Phosphotungstic Acid

Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrPSc using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrPC, while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrPC

Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin

Disease-related PrPSc [pathogenic PrP (prion protein)] is classically distinguished from its normal cellular precursor, PrPC(cellular PrP) by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease-resistant fragments of PrPSc using PK (proteinase K), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrPSc in its full-length form. In the present study, we show that thermolysin can degrade PrPC while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML (Rocky Mountain Laboratory) prions, the majority of PK-sensitive disease-related PrP isoforms do not appear to contribute significantly to infectivity. In vCJD (variant Creutzfeldt–Jakob disease), the human counterpart of BSE (bovine spongiform encephalopathy), up to 90% of total PrP present in the brain resists degradation with thermolysin, whereas only ∼15% of this material resists digestion by PK. Detection of PK-sensitive isoforms of disease-related PrP using thermolysin should be useful for improving diagnostic sensitivity in human prion diseases

Etude du rôle biologique de la PrP cellulaire et pathologique dans des cultures primaires de cellules nerveuses

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Conformation-dependent membrane permeabilization by neurotoxic PrP oligomers: The role of the H2H3 oligomerization domain

International audienceThe relationship between prion propagation and the generation of neurotoxic species and clinical onset remains unclear. Several converging lines of evidence suggest that interactions with lipids promote various precursors to form aggregation-prone states that are involved in amyloid fibrils. Here, we compared the cytotoxicities of different soluble isolated oligomeric constructs from murine full-length PrP and from the restricted helical H2H3 domain with their effects on lipid vesicles. The helical H2H3 domain is suggested to be the minimal region of PrP involved in the oligomerization process. The discrete PrP oligomers of both the full-length sequence and the H2H3 domain have de novo beta-sheeted structure when interacting with the membrane. They were shown to permeabilize synthetic negatively charged vesicles in a dose-dependent manner. Restricting the polymerization domain of the full-length PrP to the H2H3 helices strongly diminished the ability of the corresponding oligomers to associate with the lipid vesicles. Furthermore, the membrane impairment mechanism occurs differently for the full-length PrP oligomers and the H2H3 helices, as shown by dye-release and black lipid membrane experiments. The membrane damage caused by the full-length PrP oligomers is correlated to their neuronal toxicity at submicromolar concentrations, as shown by cell culture assays. Although oligomers of synthetic H2H3 could compromise in vitro cell homeostasis, they followed a membrane-disruptive pattern that was different from the full-length oligomers, as revealed by the role of PrPC in cell viability assays

Prion Strain- and Species-Dependent Effects of Antiprion Molecules in Primary Neuronal Cultures▿

Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds. Here, we report that prion strains from different species can propagate in primary neuronal cultures derived from transgenic mouse lines overexpressing ovine, murine, hamster, or human prion protein. Using this newly developed cell system, the activity of three generic compounds known to cure prion-infected cell lines was evaluated. We show that the antiprion activity observed in neuronal cultures is species or strain dependent and recapitulates to some extent the activity reported in vivo in rodent models. Therefore, infected primary neuronal cultures may be a relevant system in which to investigate the efficacy and mode of action of antiprion drugs, including toward human transmissible spongiform encephalopathy agents

Endogenous PrP conversion is required for prion-induced neuritic alterations and neuronal death

International audienceBackground: Mechanisms involved in prion induced neuronal death are still enigmatic. In vivo, both neurons and astrocytes support prion propagation leading to abnormal production of a misfolded protein called scrapie prion protein (PrPSc). Although crucial for prion replication and accumulation of aggregated PrPSc, the role of the normal form of prion protein,the cellular PrP (PrPC), is still controversial in prion-induced neurotoxicity. Objectives : In this study we investigated the role of neuronal PrPC and the contribution of astrocytes in prion-induced neuronal death. Methods: We have previously shown that prions could be efficiently propagated in primarily cultured neurons and astrocytes (Cronier et al. 2004). Here, we have set up a model in which neurons devoid of PrPC or expressing PrPC from different species are cocultured with prion-infected astrocytes that continuously deliver physiological concentrations of PrPSc. Results : In these conditions, we showed that scrapie-infected astrocytes exacerbate prion-induced cell death solely in neurons expressing transconformable PrPC. Although infectious, conditioned medium of prion-infected astrocytes did not display any acute neuronal toxicity. However PrPSc accumulation in neurons led to neuritic damage and cell death associated with an increased sensitivity to glutamate and reactive oxygen species. Discussion : Our results indicate that interaction between neuronal PrPC and PrPSc is not sufficient to induce neuronal death but that PrPC transconformation is required for prion-associated neurotoxicity. In addition, it is unlikely that a dysfunction of prion-infected astrocytes is involved in the initiation of neuronal death process induced by prions. Altogether, our findings support the view that prion infection and subsequent PrPC transconformation trigger impairment of neuronal homeostasis by sensitizing neurons to environmental stress that are regulated by neighbouring cells including astrocyte

Endogenous PrP conversion is required for prion-induced neuritic alterations and neuronal death

Background: Mechanisms involved in prion induced neuronal death are still enigmatic. In vivo, both neurons and astrocytes support prion propagation leading to abnormal production of a misfolded protein called scrapie prion protein (PrPSc). Although crucial for prion replication and accumulation of aggregated PrPSc, the role of the normal form of prion protein,the cellular PrP (PrPC), is still controversial in prion-induced neurotoxicity. Objectives : In this study we investigated the role of neuronal PrPC and the contribution of astrocytes in prion-induced neuronal death. Methods: We have previously shown that prions could be efficiently propagated in primarily cultured neurons and astrocytes (Cronier et al. 2004). Here, we have set up a model in which neurons devoid of PrPC or expressing PrPC from different species are cocultured with prion-infected astrocytes that continuously deliver physiological concentrations of PrPSc. Results : In these conditions, we showed that scrapie-infected astrocytes exacerbate prion-induced cell death solely in neurons expressing transconformable PrPC. Although infectious, conditioned medium of prion-infected astrocytes did not display any acute neuronal toxicity. However PrPSc accumulation in neurons led to neuritic damage and cell death associated with an increased sensitivity to glutamate and reactive oxygen species. Discussion : Our results indicate that interaction between neuronal PrPC and PrPSc is not sufficient to induce neuronal death but that PrPC transconformation is required for prion-associated neurotoxicity. In addition, it is unlikely that a dysfunction of prion-infected astrocytes is involved in the initiation of neuronal death process induced by prions. Altogether, our findings support the view that prion infection and subsequent PrPC transconformation trigger impairment of neuronal homeostasis by sensitizing neurons to environmental stress that are regulated by neighbouring cells including astrocyte

Endogenous prion protein conversion is required for prion-induced neuritic alterations and neuronal death

International audiencePrions cause fatal neurodegenerative conditions and result from the conversion of host-encoded cellular prion protein (PrPC) into abnormally folded scrapie PrP (PrPSc). Prions can propagate both in neurons and astrocytes, yet neurotoxicity mechanisms remain unclear. Recently, PrPC was proposed to mediate neurotoxic signaling of beta-sheet-rich PrP and non-PrP conformers independently of conversion. To investigate the role of astrocytes and neuronal PrPC in prion-induced neurodegeneration, we set up neuron and astrocyte primary cocultures derived from PrP transgenic mice. In this system, prion-infected astrocytes delivered ovine PrPSc to neurons lacking PrPC (prion-resistant), or expressing a PrPC convertible (sheep) or not (mouse, human). We show that interaction between neuronal PrPC and exogenous PrPSc was not sufficient to induce neuronal death but that efficient PrPC conversion was required for prion-associated neurotoxicity. Prion-infected astrocytes markedly accelerated neurodegeneration in homologous cocultures compared to infected single neuronal cultures, despite no detectable neurotoxin release. Finally, PrPSc accumulation in neurons led to neuritic damages and cell death, both potentiated by glutamate and reactive oxygen species. Thus, conversion of neuronal PrPC rather than PrPC-mediated neurotoxic signaling appears as the main culprit in prion-induced neurodegeneration. We suggest that active prion replication in neurons sensitizes them to environmental stress regulated by neighboring cells, including astrocytes.-Cronier, S., Carimalo, J., Schaeffer, B., Jaumain, E., Beringue, V., Miquel, M.-C., Laude, H., Peyrin, J.-M. Endogenous prion protein conversion is required for prion-induced neuritic alterations and neuronal death. FASEB J. 26, 3854-3861 (2012). www.fasebj.or

RML prion infectivity following digestion with 1 mg/ml pronase E.

<p>10% (w/v) RML brain homogenate was incubated with pronase E (1 mg/ml at 37°C) for varying incubation times. For each time point RML prion infectivity was measured by the Scrapie Cell Assay and expressed as a percentage of total infectivity present in the untreated sample; mean ± S.E.M. (<i>n</i> = 5).</p