LACK OF CELLULAR PRION PROTEIN UNMASKS NMDA NR2D SUBUNIT RECEPTOR FUNCTION WITH CONSEQUENCES TOWARD SYNAPTIC TRANSMISSION AND EXCITOTOXICITY
Authors: Khosravani H , Zhang Y , Tsutsui S , Hameed S , Altier C , Hamid J , Chen L , Villemaire M , Ali Z , Jirik F R , Zamponi G W 
Published: May 2017

Background: The physiological functions of endogenous cellular prion protein (PrPC)is incompletely understood. Previously, it has been shown that PrP-null mice exhibit reduced long-term (synaptic) potentiation and greater susceptibility to seizure mortality in several in vivo models of epilepsy. In addition, PrP-null neurons in culture exhibit greater excito toxic cell death in response to kainic acid exposure, and in several models of oxidative stress. Although PrP seems toplay a protective role against various forms of cellular insults, the precise mechanism of such action is unknown.

Methods: We investigated the synaptic properties of WT and PrP-null mice using cultured neurons and also brain slices from adult mice. Synaptic activity was assessed using whole-cell voltage clamp. We recorded spontaneous and evoked synaptic potentials. Extracellular field recordings of brain slices were also performed. Pharmacological agents were used to isolate all components of glutamatergic and GABA(A) mediated synaptic transmission. In addition, weassessed the effect of NMDA excitotoxicity in WT and PrP-null neurons using in vitro and in vivo experiments. We also used immunostaining, coimmunoprecipitation, and protein expression studies to quantify the relation between NMDA subtype expression and localization relative to native PrP.

Results: Recordings in the CA1 layer of adult hippocampal slices showed thatPrP-null mice exhibit a reduced threshold to evoked responses, exhibited basal hyperexcitability, and in a model of zero-Mg2+ seizures also showed lower seizure threshold. No differences were observed in paired-pulse facilitation relative to WT animals. Recordings from mature hippocampal cultures showed slightly altered AMPA and GABAA miniature synaptic currents. NMDA mEPSCs were observed to be increased in amplitude and significantly prolonged in decaytime. NMDA-evoked currents also exhibited markedly prolonged deactivation kinetics. This effect on evoked NMDA currents was reproduced in WT neurons byindependent PrP-RNAi, NR2D-RNAi transfection, and eliminated by PrPCtransfection into PrP-null neurons. In addition, PrP coimmunoprecipitated with NR2D and not NR2B NMDA receptor subunits. In vitro and in vivo experiments utilizing transient exposure to NMDA showed greater cell death in PrP-nullneurons, which was significantly reduced by application of an NMDA receptor antagonist.

Conclusions: These data suggest that enhanced NMDA activity is present in PrP-null neurons. Consistent with this finding, in vitro and in vivo excitotoxicity assays demonstrated increased neuronal cell death in PrP-null cultures and animals upon transient exposure to NMDA. This was confirmed at the level of synaptic currents showing prolonged receptor deactivation kinetics that were most consistent with functional activation of NR2D NMDA receptor subunits. Enhanced NMDA receptor function was paralleled by increased excitotoxicity in PrP-null mice. Our findings demonstrate a novel functional role for PrP as a modulator of synaptic NMDA currents and attributes a neuroprotective function to PrP.