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.
Highlights
Prion diseases are transmissible neurodegenerative disorders with human phenotypes including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-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]
Employing an electrophysiology model, measuring the capacity of brain homogenates derived from across the disease time-course to impair CA1 region long-term potentiation (LTP) and post-tetanic potentiation (PTP) in hippocampal slices, we observed that synaptotoxic species were present from 30% of the terminal stage of disease (TSD)
Size fractionation chromatography revealed that acute synpatotoxicity correlated with predominance of oligomeric PrP species in infected brains across all time points, while additional characterisation of cM1000 demonstrated that the predominant synaptotoxic PrPSc species up to and including 70% of the TSD were quite proteinase-sensitive
Summary
Prion diseases are transmissible neurodegenerative disorders with human phenotypes including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-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]. Experimental approaches exploiting successful rodent-adaptation of human and animal prion diseases such as CJD and scrapie have facilitated our understanding of the pathogenic evolution of these disorders [14,15,16], consensus around some fundamental aspects of pathogenesis has not been achieved. Reports of electrophysiological [18], morphological [19] and behavioural [20] disturbances prior to the mid-incubation point, generally coinciding with the first detection of
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