Highly neurotoxic monomeric α-helical prion protein

Abstract

Prion diseases are infectious and belong to the group of protein misfolding neurodegenerative diseases. In these diseases, neuronal dysfunction and death are caused by the neuronal toxicity of a particular misfolded form of their cognate protein. The ability to specifically target the toxic protein conformer or the neuronal death pathway would provide powerful therapeutic approaches to these diseases. The neurotoxic forms of the prion protein (PrP) have yet to be defined but there is evidence suggesting that at least some of them differ from infectious PrP (PrP Sc ). Herein, without making an assumption about size or conformation, we searched for toxic forms of recombinant PrP after dilution refolding, size fractionation, and systematic biological testing of all fractions. We found that the PrP species most neurotoxic in vitro and in vivo (toxic PrP, TPrP) is a monomeric, highly α-helical form of PrP. TPrP caused autophagy, apoptosis, and a molecular signature remarkably similar to that observed in the brains of prion-infected animals. Interestingly, highly α-helical intermediates have been described for other amyloidogenic proteins but their biological significance remains to be established. We provide unique experimental evidence that a monomeric α-helical form of an amyloidogenic protein represents a cytotoxic species. Although toxic PrP has yet to be purified from prion-infected brains, TPrP might be the equivalent of one highly neurotoxic PrP species generated during prion replication. Because TPrP is a misfolded, highly neurotoxic form of PrP reproducing several features of prion-induced neuronal death, it constitutes a useful model to study PrP-induced neurodegenerative mechanisms.

Cytosolic PrP Can Participate in Prion-Mediated Toxicity

“…While the mechanism of prion toxicity remains to be defined, it is established that PrP expression is required for susceptibility to the neurotoxic agent. The essential requirement for PrP expression in prion-induced neurotoxicity may suggest an intermediate in the conversion of PrPC to PrPSc is the neurotoxic agent (60,61). An alternative possibility is that neurotoxicity results from PrPSc interference with the normal biosynthesis and metabolism of PrPC (25).…”

Prion Infectivity Plateaus and Conversion to Symptomatic Disease Originate from Falling Precursor Levels and Increased Levels of Oligomeric PrP Sc Species

“…2 and 4 and Table 1) differed from endpoint samples only in quantity and not quality. A toxic monomeric alpha-helical form of PrP ("TPrP") has been deduced from refolding unglycosylated recombinant PrP and assay upon the PK-1 subline of N2a neuroblastoma cells (27). Toxicity mediated by TPrP was observed in the range of 0.5 to 5 g/ml (27,28), which is in the same general range as PrP C levels measured here in brain homogenates of Prnp ϩ/ϩ and Prnp 0/ϩ mice (i.e., ϳ1.1 and 0.4 g/ml, respectively; Fig.…”

“…A calibration curve was prepared with either recombinant PrP(23-231, 129M) for samples containing full-length PrP Sc or recombinant PrP(90-231, 129M) for samples containing truncated rPrP Sc [PrP (27)(28)(29)(30)] after proteinase K (PK) treatment. The PK-untreated sample containing PrP was divided into a native and a denatured aliquot, where the latter was denatured with 4 M guanidinium HCl for 5 min at 80°C.…”

Single-molecule approaches to prion protein misfolding

“…Second, we found no evidence for a stable misfolded form of monomeric PrP, as proposed recently. 42 Since measurements like those in Figure 3 are in equilibrium, all possible structures are explored during the trajectories, but PrP C was the only stable, long-lived structure observed. 26 Hence any alternate structure must either require the truncated residues 23-90 or else specific experimental conditions not found in the single-molecule measurements.…”