Prion Protein Is a Key Determinant of Alcohol Sensitivity through the Modulation of N-Methyl-D-Aspartate Receptor (NMDAR) Activity

The prion protein (PrP) is absolutely required for the development of prion diseases; nevertheless, its physiological functions in the central nervous system remain elusive. Using a combination of behavioral, electrophysiological and biochemical approaches in transgenic mouse models, we provide strong evidence for a crucial role of PrP in alcohol sensitivity. Indeed, PrP knock out (PrP−/−) mice presented a greater sensitivity to the sedative effects of EtOH compared to wild-type (wt) control mice. Conversely, compared to wt mice, those over-expressing mouse, human or hamster PrP genes presented a relative insensitivity to ethanol-induced sedation. An acute tolerance (i.e. reversion) to ethanol inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated excitatory post-synaptic potentials in hippocampal slices developed slower in PrP−/− mice than in wt mice. We show that PrP is required to induce acute tolerance to ethanol by activating a Src-protein tyrosine kinase-dependent intracellular signaling pathway. In an attempt to decipher the molecular mechanisms underlying PrP-dependent ethanol effect, we looked for changes in lipid raft features in hippocampus of ethanol-treated wt mice compared to PrP−/− mice. Ethanol induced rapid and transient changes of buoyancy of lipid raft-associated proteins in hippocampus of wt but not PrP−/− mice suggesting a possible mechanistic link for PrP-dependent signal transduction. Together, our results reveal a hitherto unknown physiological role of PrP on the regulation of NMDAR activity and highlight its crucial role in synaptic functions

Expression profiles of prion and doppel proteins and of their receptors in mouse splenocytes.

Doppel (Dpl) shares common structural features with the prion protein (PrP) whose pathologic isoform is considered as the causative agent of prion diseases. Although their physiological functions in the immune system remain largely unknown, we demonstrated that substantial amounts of PrP and Dpl are expressed by spleen cells notably B lymphocytes, granulocytes and DC, but not T lymphocytes and NK. To characterize trans-interacting partners of PrP and Dpl on mouse splenocytes, fluorescent PrP and Dpl tetramers were produced and used as tracers. Both tetramers specifically bind to B lymphocytes, dendritic cells, macrophages and granulocytes and in a lesser extend to T lymphocytes. No binding was observed on NK, follicular dendritic cells and mesenchymal spleen cells. The activation of intracellular transduction signals (i.e. intracellular calcium concentration and activation of the MAP kinase pathway) suggested that PrP and Dpl tetramers bind to functional receptors on B cells. None of the previously described PrP partners account for the binding sites characterized here. Our study suggests a possible role for PrP and Dpl in the cell-cell interactions in the immune system

EtOH induced changes in the buoyancy of lipid-raft associated proteins.

<p>Lipid-rafts were prepared by sucrose-gradient centrifugation from hippocampus of saline (Ctrl) and EtOH-treated wt (<b>A</b>) and PrP^−/− (<b>B</b>) mice (5, 20 min post-EtOH <i>i.p</i> injection). An aliquot of each fraction was analysed by SDS-PAGE and blots were probed with the indicated antibodies. One representative of four independent experiments is shown in panels <b>A</b> and <b>B</b>. Amounts of immunoreactivity of PrP, p-Y1472GluN2B, p-Y416Src-PTK and flotilin-1 were determined in fractions 4 and 5 and expressed as the percent of total immunoreactivity measured in all fractions for each protein of interest. Results are representative of 2 mice/condition in 4 independent experiments and presented as mean ± SD (N = 4; Dunnett's <i>post hoc</i>; EtOH injections versus saline injections: *<i>p</i><0.05; **<i>p</i><0.01).</p

Effect of acute exposure to EtOH on GluN2B-containing NMDARfunction in hippocampal slices of PrP^−/− and wt mice.

<p>Panel <b>A</b> represents typical traces of: NMDAR fEPSP recorded in the presence of CNQX of wt (<i>left</i>) and PrP^−/− (<i>right</i>) hippocampal slices (average of 6 single sweeps) obtained before (baseline, black), during EtOH application for 12 min (red), 10 min after EtOH wash out (grey) or in the presence of the NMDAR antagonist, APV (blue). Time course for normalized maximal peaks (<b>B</b>) for fEPSPs in the hippocampus of PrP^−/− (open) and wt (grey) mice measured before and during the bath application of 100 mM EtOH. Data are presented as mean ±SEM, percentage of baseline. Curves were significantly different from times 3–25 min using exact and Monte Carlo resampling for the Wilcoxon-Mann-Whitney test (p<0.05). Histograms show the comparison of fEPSPs peaks (<b>C</b>) and slopes (<b>D</b>) during EtOH application (0–12 min) and after EtOH wash out (17−27 min in <b>B</b>). Mean ±SEM, N  =  66, 33 in <b>C</b> for wt and PrP^−/− respectively. Comparison of fEPSPs peaks (<b>E</b>) and slopes (<b>F</b>) in wt mouse during 15 min of EtOH application and after wash out with and without 5 µM ifenprodil. Mean ± s.e.m, n = 33, 66 with and without ifenprodil respectively. **<i>p</i><0.01, ***<i>p</i><0.001 using a one-way ANOVA followed by a Dunnett's <i>post hoc</i> analysis.</p

Co-localization of PrP, fyn, GluN2B and their phosphorylated forms in hippocampal neurons in primary culture: Effects of an acute exposure to EtOH on GluN2B phosphorylation level.

<p>(A) Hippocampal neurons were co-labeled with antibodies against PrP (green, <i>left</i>) and fyn, p-Y416Src-PTK, GluN2B and p-Y1472GluN2B (red, <i>middle</i>). The merge images (<i>right</i>) correspond to the superposition of the two fluorescence signals (yellow). Nuclei were labeled with the Hoescht reagent and appeared in blue. (B) High magnification of neuronal extensions labeled with antibodies against PrP (green), p-Y1472 GluN2B (red) and PSD95 or Thy1 (blue). The merge images (<i>right</i>) correspond to the overlap of the three fluorescence signals (white). <i>Scale bar</i> = 10 µm. (C) Hippocampal neurons from wt and PrP^−/− mouse embryos were incubated with saline solution (Ctrl) or 100 mM EtOH (in NaCl 0.9%) for the indicated times. Alternatively, PP2 was pre-incubated 15 min before the addition of EtOH. Neurons were washed twice with PBS, lysed and prepared for SDS-PAGE and western blotting analysis. Blots were probed with the antibodies directed against p-Y1472GluN2B, p-Y416Src-PTK and actin to ensure correct protein loading in the different conditions. One representative of four independent experiments is shown.</p

Altered sedative effect of EtOH in PrP^−/−, PrP-over-expressing mice: relation to NMDAR function.

<p>EtOH sensitivity was evaluated by measuring the duration of LORR after <i>i.p</i>. injection of 2.4 (<b>A</b>, <i>left</i>) or 3.2 g/kg EtOH (<b>A</b>, <i>right</i> and <b>B</b>, <b>C</b>, <b>D, E</b>). In panel <b>E</b>, the EtOH injection was preceded by an <i>i.p</i>. injection of 0.2 mg/kg of the NMDAR antagonist, MK-801 or 3 mg/kg of ifenprodil. In panel <b>F</b>, sedation induced by GABA modulators was evaluated by measuring the duration of LORR after <i>i.p</i>. injection of 50 mg/kg diazepam (<i>left</i>) or 30 g/kg THIP (<i>right</i>). For LORR experiments (panels <b>A</b>, <b>C</b>, <b>D</b>, <b>E</b> and <b>F</b>), each symbol represents a mouse; the number of mice tested is shown below each condition; the dark line is the median value. The wt mice i.e. C57BL6/J x 129/Sv for panels <b>A, B, C, E</b> and <b>F</b> or C57BL6/J for panel <b>D</b> (noted Wt_B6) were taken as controls. Significance (p) was calculated using the nonparametric Mann-Whitney test; *p<0.05; **p<0.01; ***p<0.001; n.s; not significant. Blood EtOH concentration (<b>B</b>) was performed on four mice of each genotype for each time point and represented as the mean ± SD (<i>student t</i> test).</p