Date Published: August 8, 2018
Publisher: Public Library of Science
Author(s): Simote Totauhelotu Foliaki, Victoria Lewis, David Isaac Finkelstein, Victoria Lawson, Harold Arthur Coleman, Matteo Senesi, Abu Mohammed Taufiqual Islam, Feng Chen, Shannon Sarros, Blaine Roberts, Paul Anthony Adlard, Steven John Collins, David Westaway.
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) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies.
Prion diseases constitute a group of transmissible neurodegenerative disorders with the spectrum encompassing several human phenotypes, the most common being Creutzfeldt-Jakob disease (CJD), as well as a number of animal diseases including bovine spongiform encephalopathy (“mad cow” disease) and scrapie in sheep [1, 2]. Regardless of disease phenotype, misfolding of PrPC into disease-associated conformers (herein collectively designated PrPSc), with their subsequent aggregation and accumulation, appears critical to pathogenesis although the precise neurotoxic species and how such species provoke neuronal dysfunction and loss leading to the onset of clinical illness remain unresolved. The precise composition of the infectious unit or “prion” also remains to be determined, although considerable evidence supports that PrPSc is the major, if not exclusive, component (the “protein only” hypothesis) . Historically, PrPSc has been considered to be highly protease-resistant (designated PrPres after protease treatment) but recent evidence supports the existence of a broader spectrum, including protease-sensitive conformers, which most likely contribute to pathogenesis and may comprise up to 90% of misfolded prion protein in diseased brains [4, 5].
Herein we report the acute synaptotoxicity of ex vivo, prion-containing preparations and provide important new molecular pathophysiological insights. Utilising a number of experimental approaches, our findings revealed that: (1) PrPSc (particularly at least modestly PK-resistant isoforms most likely as multimeric species) is directly synaptotoxic; (2) that the acute synaptotoxicity was similar across the two prion strains examined and appears independent of tissue source of prions when balanced for PrPSc levels; (3) the acute synaptotoxicity is not reliant on hippocampal PrPC expression albeit with noteworthy molecular and electrophysiological differences in the presence or absence of PrPC; and (4) both pre- and post-synaptic functions are deleteriously affected, with the possibility of enhanced vulnerability of the former compartment.