Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species

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.

Electrophysiological Investigations of Prion Protein Roles in Health and Disease

“…This impairment is associated with memory and behavioral deficits [27]. However, this is still a point of contention as normal LTP within these regions has also been observed and the discrepancy appears un-related to the genetic background of the mouse model [5,[28][29][30]. Hippocampal CA1 metaplasticity is also impaired in PrP knockout mice; here the post-synaptic responses become enhanced as opposed to being depressed when a low frequency stimulation is paired with a couple trains of theta-burst stimulation (consists of 10 trains delivered at 5 Hz where each train comprises of 4 pulses delivered at 100 Hz) at an interval of ~25 minutes [29].…”

“…Another way of studying the specific effects of exposure to toxic PrPD is through assessing the acute response of cells [5,64]. A recent study has reported that acute exposure of ex vivo hippocampal slices to ex vivo mouse-adapted human prions (M1000 and MU02) disrupts CA1 LTP by causing dysfunctions of both preand postsynaptic activity [5].…”

“…These include the short-term plasticity (STP), long-term potentiation (LTP), and long-term depression (LTD) [4]. STP is usually detected as a rapid physiological alteration of neuronal activity that lasts milliseconds to seconds immediately after neurons receiving a stimulation [5,6]. This short-term function is usually due to a rapid enhanced release of neurotransmitters that overwhelmingly activate the preexisting receptors on the post-synaptic terminals [5,6].…”

“…STP is usually detected as a rapid physiological alteration of neuronal activity that lasts milliseconds to seconds immediately after neurons receiving a stimulation [5,6]. This short-term function is usually due to a rapid enhanced release of neurotransmitters that overwhelmingly activate the preexisting receptors on the post-synaptic terminals [5,6]. Through such a mechanism, STP can be evoked by a paired-stimulus delivered a few milliseconds apart, in which the post-synaptic response evoked by the second stimulus indicates the probability of neurotransmitter release at a synapse or a population of synapses [5,6].…”

“…This short-term function is usually due to a rapid enhanced release of neurotransmitters that overwhelmingly activate the preexisting receptors on the post-synaptic terminals [5,6]. Through such a mechanism, STP can be evoked by a paired-stimulus delivered a few milliseconds apart, in which the post-synaptic response evoked by the second stimulus indicates the probability of neurotransmitter release at a synapse or a population of synapses [5,6]. Further, with repetitive short (~0.5-1 min) trains of high frequency (~100-200 Hz) stimulation, neurotransmitters are released at a substantial level, which extensively depolarizes the post-synaptic membrane causing activation of the NMDA receptor (NMDAR), a glutamate receptor that modulates the post-synaptic currents when it is active [7].…”