Fig. 3

In vivo P2X7R inhibition reverts the UPS dysfunction that P301S mice mimic from AD patients. A Representative Western blot and quantification of P2X7R (A) and polyubiquitin proteins (B) using homogenates from hippocampal necropsies from Alzheimer’s disease (AD) patients and non-affected individuals (CTL). Graphs show the quantification of P2X7R (A) or polyubiquitin (B) protein expression in hippocampal homogenates from AD (n = 8) and control (n = 5). α-tubulin (α-Tub) or β-actine expression was used as loading control for normalization purposes. 100% value corresponds to the averaged amount of P2X7 or polyubiquitinated proteins (polyUb) detected in CTL. C Representative hippocampal sections from AD patients stained with polyUb marker (FK2, red channel), P2X7R (green channel), and DAPI (gray channel). Merged images are also shown. Arrowheads indicate neural cells expressing high levels both of P2X7R and polyUb. Scale bar: 50 μm. D Western blot and quantification of β5 and β1 protein levels in homogenates from hippocampal AD patients (n = 8) and control individuals (n = 4). Quantification of β5 mRNA (PSMB5) (E) and β1 mRNA (PSMB6) (F) in the hippocampus from human controls (n = 4) and AD patients (n ≥ 7). In all cases, the 100% value corresponds to the averaged amount of protein or messenger of β5 and β1 detected in the hippocampi of non-affected individuals. G Representative Western blot image and quantification of Nfr2 transcription factor both in human controls (n = 3) and AD patients (n = 5). Data in bar graphs represent mean ± s.e.m. *p ≤ 0.05; **p ≤ 0.01; **** P ≤ 0.0001 using an unpaired two-tailed Student’s t-test. H Representative immunoblot of P2X7R protein levels in hippocampal homogenates from 9-month-old P301S and wild-type (WT) mice. -tubulin (α-Tub) was used as a loading control. Graph shows P2X7R protein levels in P301S and WT mice (n = 5 mice per genotype). I Western blot and quantification of polyubiquitinated proteins using hippocampal homogenates from 9-month-old wild-type (WT) mice and P301S mice treated with Vehicle (Veh) or GSK 1482160A (GSK) for 3 weeks. GAPDH was used as a loading control. 100% value corresponds to the averaged amount of polyubiquitinated proteins (polyUb) detected in Veh-WT mice (n = 4–6 per genotype and condition). J Representative images of hippocampal slices stained with FK2 antibody labeling polyubiquitinated proteins in Veh- or GSK-treated P301S mice. Scale bar: 100 μm. Graph shows the number of hippocampal cells bearing intracellularly polyubiquitinated aggreges in Veh- or GSK-treated P301S mice (n = 4–6 per genotype). *** P ≤ 0.001 using an unpaired two-tailed Student’s t-test. K) Western blot and quantification of β5 and β1 protein levels in hippocampal homogenates from Veh-WT mice, Veh-P301S, and GSK-P301S mice (≥ 5 mice per treatment and genotype). Quantification of β5 mRNA (psmb5) (L) and β1 mRNA (psmb6) (M) in the hippocampus from Veh-WT mice, Veh-P301S and GSK-P301S (n ≥ 5 per genotype and treatment). In all cases, the 100% value corresponds to the averaged amount of protein or messenger of β5 and β1 detected in the hippocampi of Veh-WT mice. *p ≤ 0.05; **p ≤ 0.01; *** P ≤ 0.001 using a one-way ANOVA followed by Tukey’s post hoc test. N Representative Western blot image and quantification of Nfr2 transcription factor in the hippocampus from Veh-WT mice, Veh-P301S, and GSK-P301S (n ≥ 4 per genotype and treatment). 100% value corresponds to the average amount of Nrf2 detected in the hippocampi of Veh-WT or GSK-P301S mice. Data in bar graphs represent mean ± s.e.m. *p ≤ 0.05; **p ≤ 0.01; using an unpaired two-tailed Student’s t-test