Increased Proportions of C1 Truncated Prion Protein Protect Against Cellular M1000 Prion Infection (original) (raw)
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Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species
PLoS pathogens, 2018
Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) r...
Prion Protein Misfolding, Strains, and Neurotoxicity: An Update from Studies on Mammalian Prions
International Journal of Cell Biology, 2013
Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders affecting humans and other mammalian species. The central event in TSE pathogenesis is the conformational conversion of the cellular prion protein, PrP C , into the aggregate, -sheet rich, amyloidogenic form, PrP Sc . Increasing evidence indicates that distinct PrP Sc conformers, forming distinct ordered aggregates, can encipher the phenotypic TSE variants related to prion strains. Prion strains are TSE isolates that, after inoculation into syngenic hosts, cause disease with distinct characteristics, such as incubation period, pattern of PrP Sc distribution, and regional severity of histopathological changes in the brain. In analogy with other amyloid forming proteins, PrP Sc toxicity is thought to derive from the existence of various intermediate structures prior to the amyloid fiber formation and/or their specific interaction with membranes. The latter appears particularly relevant for the pathogenesis of TSEs associated with GPI-anchored PrP Sc , which involves major cellular membrane distortions in neurons. In this review, we update the current knowledge on the molecular mechanisms underlying three fundamental aspects of the basic biology of prions such as the putative mechanism of prion protein conversion to the pathogenic form PrP Sc and its propagation, the molecular basis of prion strains, and the mechanism of induced neurotoxicity by PrP Sc aggregates.
Molecular Distinction between Pathogenic and Infectious Properties of the Prion Protein
Journal of Virology, 2003
Tg(PG14) mice express a prion protein (PrP) with a nine-octapeptide insertion associated with a human familial prion disease. These animals spontaneously develop a fatal neurodegenerative disorder characterized by ataxia, neuronal apoptosis, and accumulation in the brain of an aggregated and weakly protease-resistant form of mutant PrP (designated PG14 spon ). Brain homogenates from Tg(PG14) mice fail to transmit disease after intracerebral inoculation into recipient mice, indicating that PG14 spon , although pathogenic, is distinct from PrP Sc , the infectious form of PrP. In contrast, inoculation of Tg(PG14) mice with exogenous prions of the RML strain induces accumulation of PG14 RML , a PrP Sc form of the mutant protein that is infectious and highly protease resistant. Like PrP Sc , both PG14 spon and PG14 RML display conformationally masked epitopes in the central and octapeptide repeat regions. However, these two forms differ profoundly in their oligomeric states, with PG14 RML aggregates being much larger and more resistant to dissociation. Our analysis provides new molecular insight into an emerging puzzle in prion biology, the discrepancy between the infectious and neurotoxic properties of PrP.
Journal of Biological Chemistry, 2010
The abnormally folded form of the prion protein (PrP Sc) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminaltruncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP Sc N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP Sc accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP Sc proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP Sc fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP Sc and cell pathogenesis of prion infection. Prions are the infectious agent of transmissible spongiform encephalopathies (TSE), 4 a group of fatal neurodegenerative disorders that include scrapie in sheep, bovine spongiform encephalopathie in cattle, and Creutzfeldt-Jakob disease in humans. The
Journal of Biological Chemistry, 2005
Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.
Nature Communications, 2014
Prions are lethal infectious agents thought to consist of multi-chain forms (PrPSc) of misfolded cellular prion protein (PrPC). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrPC expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrPC expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrPSc at a rate proportional to PrPC concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrPC concentration not being ra...
Rapid ex vivo reverse genetics identifies the essential determinants of prion protein toxicity
The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208-H140 hydrogen bond (“H-latch”) between the α2-α3 and β2-α2 loops of PrPC. Importantly, GDL that suppress the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced nineteen individual PrPC variants to PrPC-deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K27 or R27) within PrPC. Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-t...