Fig. 2

In vivo P2X7R activation decreases proteasomal chymotrypsin-like and post-glutamyl-like activities in neurons and microglia cells by reducing the proteasomal subunits β5 and β1 translation and transcription via Akt/GSK3/Nfr2. Chymotrypsin-like (A and D) and postglutamyl (B and E) activities were measured in hippocampal extracts from WT mice (A and B, white bars) or P2X7R^−/− mice (D and E, gray bars) treated with a single injection of 2µL 30 mM BzATP or vehicle solution (Veh) via i.c.v. for 24 h (n ≥ 4 in triplicated). 100% value corresponds to activity detected in vehicle-treated mice. In all reactions, the specificity of the fluorogenic reaction was verified by the addition of the proteasome inhibitor MG132. Representative images of Western blot using hippocampal samples from WT mice (C) or P2X7R^−/− mice (F) treated as above indicated and stained with antibodies anti-β5 and anti-β1 subunits, anti-phosphorylated (pAkt) or anti-total Akt, anti-phosphorylated (pGSK3) or anti-total GSK3, anti-Nfr2, and anti -tubulin. Graphs show the quantification of proteasome catalytic subunits β5 and β1, as well the levels of p-Akt, p-GSK3, and Nfr2 in WT mice (left side, white bars) and P2X7^−/− (right side, gray bars) mice. Membranes are probed with an anti--tubulin antibody to correct for any possible deviation on protein loading. 100% value corresponds to protein levels detected in vehicle-treated WT or P2X7^−/− mice respectively. Data in bar graphs represent mean ± s.e.m. *p ≤ 0.05; **p ≤ 0.01; *** P ≤ 0.001; using an unpaired two-tailed Student’s t-test. G Representative images of hippocampal CA3 area from UbGFP mice 24 h after being treated with a single injection via i.c.v. of 2µL 30 mM BzATP or vehicle solution and stained with antibody against GFP (green channel) and nuclear marker DAPI (blue channel). Original scale bars, 100µm. Quantification of hippocampal CA3 green cells per slice (n ≥ 5 mice per treatment and n ≥ 3 sections per mouse). *p ≤ 0.05; *** P ≤ 0.001; using a two-way ANOVA followed by Tukey’s post hoc test. H Hippocampal sections from BzATP- or vehicle-treated UbGFP mice stained with a specific neuronal marker (NeuN, red channel) and GFP (green channel) on the left side and microglial marker (Iba-1, red channel) and GFP (green channel) on the right side. Merged images showing the DAPI channel in blue are also shown. Scale bar: 100 μm. Quantification of the GFP-positive cells identified as neurons (upper) or microglial cells (down) per hippocampal slice in BzATP- or vehicle-treated UbGFP mice (n ≥ 5 mice per treatment and n ≥ 3 sections per mouse). Data in bar graphs depict means ± s.e.m. *p ≤ 0.05; **p ≤ 0.01; using an unpaired two-tailed Student’s t-test