Research Article: Early existence and biochemical evolution characterise acutely synaptotoxic PrPSc

Date Published: April 10, 2019

Publisher: Public Library of Science

Author(s): Simote Totauhelotu Foliaki, Victoria Lewis, Abu Mohammed Taufiqual Islam, Laura Jane Ellett, Matteo Senesi, David Isaac Finkelstein, Blaine Roberts, Victoria A. Lawson, Paul Anthony Adlard, Steven John Collins, David Westaway.

http://doi.org/10.1371/journal.ppat.1007712

Abstract

Although considerable evidence supports that misfolded prion protein (PrPSc) is the principal component of “prions”, underpinning both transmissibility and neurotoxicity, clear consensus around a number of fundamental aspects of pathogenesis has not been achieved, including the time of appearance of neurotoxic species during disease evolution. Utilizing a recently reported electrophysiology paradigm, we assessed the acute synaptotoxicity of ex vivo PrPSc prepared as crude homogenates from brains of M1000 infected wild-type mice (cM1000) harvested at time-points representing 30%, 50%, 70% and 100% of the terminal stage of disease (TSD). Acute synaptotoxicity was assessed by measuring the capacity of cM1000 to impair hippocampal CA1 region long-term potentiation (LTP) and post-tetanic potentiation (PTP) in explant slices. Of particular note, cM1000 from 30% of the TSD was able to cause significant impairment of LTP and PTP, with the induced failure of LTP increasing over subsequent time-points while the capacity of cM1000 to induce PTP failure appeared maximal even at this early stage of disease progression. Evidence that the synaptotoxicity directly related to PrP species was demonstrated by the significant rescue of LTP dysfunction at each time-point through immuno-depletion of >50% of total PrP species from cM1000 preparations. Moreover, similar to our previous observations at the terminal stage of M1000 prion disease, size fractionation chromatography revealed that capacity for acute synpatotoxicity correlated with predominance of oligomeric PrP species in infected brains across all time points, with the profile appearing maximised by 50% of the TSD. Using enhanced sensitivity western blotting, modestly proteinase K (PK)-resistant PrPSc was detectable at very low levels in cM1000 at 30% of the TSD, becoming robustly detectable by 70% of the TSD at which time substantial levels of highly PK-resistant PrPSc was also evident. Further illustrating the biochemical evolution of acutely synaptotoxic species the synaptotoxicity of cM1000 from 30%, 50% and 70% of the TSD, but not at 100% TSD, was abolished by digestion of immuno-captured PrP species with mild PK treatment (5μg/ml for an hour at 37°C), demonstrating that the predominant synaptotoxic PrPSc species up to and including 70% of the TSD were proteinase-sensitive. Overall, these findings in combination with our previous assessments of transmitting prions support that synaptotoxic and infectious M1000 PrPSc species co-exist from at least 30% of the TSD, simultaneously increasing thereafter, albeit with eventual plateauing of transmitting conformers.

Partial Text

Prion diseases are transmissible neurodegenerative disorders with human phenotypes including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) and Kuru, while the principal animal diseases comprise scrapie in sheep and goats, bovine spongiform encephalopathy (“mad cow” disease) and chronic wasting disease in deer, elk and moose [1, 2]. The key pathogenic event in all prion diseases is believed to be misfolding of the normal prion protein (PrPC) into altered conformers (PrPSc) with progressive accumulation of PrPSc in the brain linked to neurotoxicity through incompletely resolved mechanisms [3–8]. PrPSc, especially that found at the terminal stage of disease (TSD: the advanced stage of disease requiring animal euthanasia) was previously construed as invariably highly protease-resistant whereas more recent evidence supports a broader spectrum encompassing a substantial proportion that are protease-sensitive [9–12], with such species evident during disease evolution [13] and terminal disease [10] most likely contributing to neurotoxicity [12].

The primary purpose of this body of work was to gain insights into the time of occurrence and biophysical properties of acutely synaptotoxic PrPSc. Employing a combination of experimental approaches the current study has demonstrated some important observations, especially: that acutely synaptotoxic PrPSc species related to M1000 prions are generated from early in disease evolution generally coinciding with the propagation of transmitting species; and that acutely synaptotoxic PrPSc species most likely constitute small oligomers, appearing to undergo significant biochemical transformation relatively late in the incubation period, particularly transition from quite protease-sensitive to at least modestly protease-resistant. The early presence of synaptotoxic conformers in combination with the relative stability of their size fractionation profile and only comparatively modest increase in absolute acute synaptotoxicity from 50% of the TSD suggests that progressive failure of neuro-protective mechanisms is likely to be an important component of the eventual transition to overt prion disease.

 

Source:

http://doi.org/10.1371/journal.ppat.1007712

 

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