Abstract
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.
Highlights
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]
We report the use of an electrophysiology paradigm that allowed us to demonstrate that at least modestly proteinase K (PK)-resistant PrPSc species from two mouse-adapted prion strains (M1000 and MU02) are directly synaptotoxic causing significant acute impairment of hippocampal CA1 region long-term potentiation (LTP)
Critical to LTP-type synaptic plasticity and episodic memory generated in the hippocampus are α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) ion channels, as well as metabotropic glutamate receptors with signal transduction mediated through pathways including calcium-regulated phosphorylated extracellular signal–regulated kinase and phosphorylated cAMP response element binding protein, which alter DNA transcription with consequent ultrastructural and receptor changes at synapses [12]
Summary
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]. The primary function of PrPC in the central nervous system remains uncertain a key role for this glycosylphosphatidylinositol-anchored glycoprotein in synaptic physiology and memory has been described [6]. Aligned to such functions, PrPC has been reported as having a predominant synaptic localisation [7], with important influences on voltage-gated calcium (Ca2+) [8] and N-methyl-D-aspartate receptor (NMDAR) ion channels [9], as well as LTP [10]. Critical to LTP-type synaptic plasticity and episodic memory generated in the hippocampus are α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) and NMDAR ion channels, as well as metabotropic glutamate receptors with signal transduction mediated through pathways including calcium-regulated phosphorylated extracellular signal–regulated kinase (pERK) and phosphorylated cAMP response element binding protein (pCREB), which alter DNA transcription with consequent ultrastructural and receptor changes at synapses [12]
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