(A) Western blot analysis of the NR2D subunit protein expression in neonatal and adult hippocampal tissue obtained from the WT and PrP-null mouse

α-Actin expression was used as a loading control. (B) NMDAR subunit surface expression as visualized by immunolabel reactivity with an antibody targeted against an extracellular (N terminus) epitope of NR2D. A punctate pattern of receptor distribution is visualized along dendritic processes. The depth of field is ∼1 μm. (C) Surface expression of NR2D relative to total cellular NR2D protein content as quantified using an ELISA assay in permeabilized (P) and nonpermeabilized (NP) cells. The number of neuronal culture samples is indicated in parentheses. Error bars represent SEM. (D) Coimmunoprecipitation of PrP and NR2D using both permutations of tag and probe showing that PrP and NR2D are in a complex. In the top panel, the blot was probed with a PrP antibody, and in the bottom panel, membrane was probed with NR2D antibody. The lane labeled control reflects beads without antibody. The experiment is a representative example of four different repetitions for both neonatal and adult mouse hippocampal tissue. (E) Western blot demonstrating the lack of coimmunoprecipitation between NR2B and PrP, whereas NR2B can be detected in brain homogenate (input). (F) Costaining of WT mouse hippocampal neurons for PrP (red) and NR2D (blue). The cells were not permeabilized, thus allowing for the selective staining of cell surface protein. The white line in the top left panel indicates the position of the linescan shown in the bottom left panel. The rectangle in the merged image (top right) corresponds to the magnified images shown at the bottom right. The arrowheads highlight examples of clear colocalization between NR2D and PrP. Bars: (B, top left) 7.5 μm; (B, top right and F, top) 10 μm; (B, bottom) 1 μm; (F, bottom) 2 μm.<p><b>Copyright information:</b></p><p>Taken from "Prion protein attenuates excitotoxicity by inhibiting NMDA receptors"</p><p></p><p>The Journal of Cell Biology 2008;181(3):551-565.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364707.</p><p></p

(A) Representative examples of raw mEPSCs recorded in mature (12–16 DIV) WT and PrP-null hippocampal neurons in culture

In PrP-null neurons, NMDA-mediated mEPSCs were observed to be larger and showed prolonged decay times. (B) Event histograms for mEPSC amplitude (top) and decay time (bottom). Note that mEPSCs in PrP-null neurons exhibit a shift toward larger amplitude events and increased decay time constants. (C) Cumulative probability plots for mEPSC amplitude and decay times showing a shift in each summed distribution toward larger events with longer decay times (P < 0.05; Kolmogorov-Smirnov test). (D) Mean values for mEPSC waveform parameters showing increased EPSC amplitudes and prolonged decay times. Here, decay time refers to the time required for an e-fold reduction in peak current amplitude. Data are represented as mean ± SEM (error bars), with statistical significance denoted as *, P < 0.05 and **, P < 0.001. Numbers in parentheses indicate the number of cells.<p><b>Copyright information:</b></p><p>Taken from "Prion protein attenuates excitotoxicity by inhibiting NMDA receptors"</p><p></p><p>The Journal of Cell Biology 2008;181(3):551-565.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364707.</p><p></p

(A) Fluoro-Jade labeling of neuronal bodies and processes in hippocampal sections in response to injection of vehicle (left) or NMDA (10-nmol equivalent; right)

(B) Quantification of lesion size relative to the hippocampus over a series of three to six sections per animal ( = 5 per experimental group). Data are represented as mean ± SEM (error bars), with statistical significance denoted as **, P < 0.001. Bar, 200 μm.<p><b>Copyright information:</b></p><p>Taken from "Prion protein attenuates excitotoxicity by inhibiting NMDA receptors"</p><p></p><p>The Journal of Cell Biology 2008;181(3):551-565.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364707.</p><p></p

(A) Paired pulses evoked by stimulation of the Schaffer collaterals in slices from P30–45 mice in normal artificial cerebrospinal fluid (aCSF)

(B) Quantification of the number of population spikes in WT and PrP-null slices in aCSF. (C, top) Minimum stimulus intensity required to evoke a single population spike in WT and PrP-null slices. (middle) Stimulus intensity required to evoke maximum single population spike amplitude. (bottom) Extent of paired pulse facilitation in WT and PrP-null slices. (D, top) Field potentials recorded from PrP-null slices before and after the application of 50 μM APV as shown for P2. (bottom) Quantification of the number of population spikes before and after APV application in PrP-null slices. (E) Evoked field potentials recorded after 5 min of perfusion in zero-magnesium aCSF (ZM-aCSF). The gray arrows indicate successive population spikes, which are augmented in the PrP-null slices. (F, left) The number of population spikes overriding the fEPSP in slices exposed to ZM-aCSF. (second graph) Time to the observance of the first seizurelike discharge in ZM-aCSF. (third graph) Time to the occurrence of the first seizurelike event (SLE) upon perfusion with ZM-aCSF. (right) Duration of seizurelike events in WT and PrP-null slices. The black and gray arrowheads indicate the primary population spikes and the additional population spikes, respectively, overriding the fEPSP in each pulse (P1 and P2); these latter polyspikes were only observed in PrP-null mice. Data are represented as mean ± SEM (error bars) with statistical significance denoted as *, P < 0.05 and **, P < 0.001. Numbers in parentheses indicate the number of slices.<p><b>Copyright information:</b></p><p>Taken from "Prion protein attenuates excitotoxicity by inhibiting NMDA receptors"</p><p></p><p>The Journal of Cell Biology 2008;181(3):551-565.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364707.</p><p></p

(A) Light microscope images of neuronal cultures after 20 min of exposure to 0

3, 0.6, and 1.0 mM NMDA followed by 24-h recovery. Cells were stained with trypan blue (dark blue) and TUNEL (brown); methyl green was used as the counterstain. (B) Mean cell counts for trypan blue– and TUNEL-stained cells in WT and PrP-null cultures. (C) Light microscope images showing TUNEL-stained neurons from PrP-null mice in the presence of NMDA and NMDA + APV. (D) Percentage of TUNEL-positive neurons from PrP-null mice in response to NMDA and NMDA + APV. The drug concentrations were 1 mM NMDA and 100 μM APV. Data are represented as mean ± SEM (error bars), with statistical significance denoted as *, P < 0.05 and **, P < 0.001. Data were obtained from four culture rounds, and six random fields were imaged per condition. Bar, 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Prion protein attenuates excitotoxicity by inhibiting NMDA receptors"</p><p></p><p>The Journal of Cell Biology 2008;181(3):551-565.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364707.</p><p></p

TcdB, but not TcdA triggers CXCL8/IL-8 production and release from Caco-2 cells in a manner dependent on extracellular nucleotides and the P2Y₆ receptor.

<p>(A) CXCL8/IL-8 release from Caco-2 cells treated with purified TcdA or TcdB (16 hr). N = 5; * denotes p<0.05 compared to TcdA. (B) TcdB, but not TcdA, treatment of Caco-2 cells increases cell death as assessed by LDH release. N = 5; * denotes p<0.05 compared to TcdA. TcdB-induced CXCL8/IL-8 release is significantly reduced by (C) MRS2578 (10 μM) and (D) co-treatment with apyrase (20 u/mL). N = 5; * denotes p<0.05 compared to no treatment; # denotes p<0.05 compared to all groups. </p

TcdA/B-induced intestinal inflammation and permeability are attenuated by inhibiting the P2y₆ receptor <i>in</i><i>vivo</i>.

<p>(A) Intrarectal instillation of TcdA/B (50 μg/100 μL for 4 hrs) triggers a significant increase in colonic tissue myeloperoxidase (MPO), an effect that is significantly reduced by pretreating mice with the P2Y₆ inhibitor MRS 2578 (100 μL of 10 μM; in PBS via intrarectal instillation). N = 6/group; * denotes p<0.05 compared to PBS vehicle groups; # denotes p<0.05 compared to TcdA/B treatment with vehicle (DMSO). (B) Pretreating mice with MRS 2578 (100 μL of 10 μM; in PBS via intrarectal instillation) inhibits TcdA/B-induced increases in permeability as assessed by FITC-flux from the colonic lumen in to the serum. N = 6/group; * denotes p<0.05 compared to PBS vehicle groups; # denotes p<0.05 compared to TcdA/B treatment with vehicle (DMSO). (C) Pretreating mice with MRS 2578 reduces the histological inflammatory score and the (D) percentage of the colonic tissue section exhibiting architectural changes (% architecture change). N = 12 sections/group; * denotes P<0.05 compared to vehicle pretreatment. (E) Representative colonic sections stained with hematoxylin and eosin from mice treated with vehicle, MRS2578 alone, vehicle + TcdA/B and MRS2578 + TcdA/B; N = 6/group.</p

<i>C. difficile</i> TcdA/B triggers the release of UDP from Caco-2 cells that express a functional P2Y₆ receptor.

<p>(A) Western blot analysis of lysates reveals the expression of the P2Y₆ receptor in differentiated Caco-2 cells and PMA-differentiated THP-1 macrophages (included as positive control). (B) Stimulation of the Caco-2 cells with the selective P2Y₆ receptor agonist 5-OMe-UDP (1 μM) increases intracellular calcium concentrations as assessed by fluorescence imaging. (B-i) Pseudocolour images of Caco-2 cells before and after 5-OMe-UDP treatment. (B-ii) Representative traces of individual cells challenged with 5-OMe-UDP. (B-iii) The mean of the 5-OMe-UDP-induced calcium responses (n=46; grey denotes the standard error of the mean). (C) P2Y₆ receptor agonist 5-OMe-UDP triggers CXCL8/IL-8 release from Caco-2 cells, an effect that blocked by the potent P2Y₆ receptor antagonist MRS2578. N = 6; * denotes p<0.05 compared to control; # denotes p<0.05 compared to vehicle; % denotes p<0.05 compared to vehicle and 1 μM MRS 2578. (D) TcdA/B triggers the release of UDP as assessed by HPLC. i – control treated culture supernatant; ii – UDP-spiked control culture supernatant (100 μM UDP); iii – TcdA/B-spiked control culture supernatant (10 μg/mL); iv – TcdA/B-treated cell culture supernatant (10 μg/mL; 16 hr). (E) Summary data from HPLC measurement of TcdA/B-induced UDP release. N=5; * denotes p<0.05.</p

TcdA/B-induced CXCL8/IL-8 production from Caco-2 IECs involves the NFκB activation, an effect that is inhibited by pharmacological blockade of the P2Y₆ receptor by MRS2578.

<p>(A) TcdA/B-induced CXCL8/IL-8 release is inhibited by pretreatment with the selective NFκB pathway inhibitor BAY 11-7085 (20 μM). N=6; ** denotes p<0.005 compared to vehicle-treated TcdA/B stimulated cells (10 μg/mL). (B) Representative western blot for phosphorylated p65 (P-p65) in lysates from TcdA/B (10 μg/mL) stimulated Caco-2 IECs over the course of 60 min in the presence of the P2Y₆ antagonist MRS2578 (10 μM) or vehicle control (DMSO). (C) The summarized western blot data for P-p65 expressed as a percentage of the total p65. N = 4, *, denotes p<0.05 compared to time 0 min; # denotes p<0.05 compared to respective vehicle control (DMSO). </p

Inhibition of the P2Y₆ receptor attenuates TcdA/B-induced intestinal epithelial barrier dysfunction in Caco-2 IECs.

<p>(A) TcdA/B-induced (10 μg/mL) FITC-flux is significantly reduced by the selective P2Y₆ receptor antagonist MRS 2578 (10 μM). N=4; * denotes p<0.05 compared to vehicle and MRS2578. # denotes p<0.05 compared to TcdA/B. (B) 5-OMe-UDP (100 μM) increases FITC-flux in Caco-2 monolayers, an effect that is significantly attenuated by MRS2578 (10 μM). N=4; * denotes p<0.05 compared to vehicle and MRS2578. # denotes p<0.05 compared to 5-OMe-UDP. (C) Apical administration of TcdA/B (10 μg/mL) or 5-OMe-UDP (100 μM) for 4 hr triggers a redistribution of ZO-1 in Caco-2 monolayers, an effect that is blocked by pretreatment with MRS 2578 (10 μM; N=4).</p