Fig 2: PTX3 does not affect the total amount of surface AMPARs AWestern blotting analysis of total and surface (biotinylated) GluA subunits of control and PTX3-treated neurons as indicated showing no differences either for the total levels of expression or for the global surface levels of the different GluA subunits.BQuantification of total receptors levels was normalized to GAPDH in each sample, whereas surface GluA subunits were normalized to surface transferrin receptor, TfR, in each sample. Data are shown as ratio PTX3 over control for each GluA subunit (total: Ctr = 1 ± 0; GluA2/3 = 0.943 ± 0.036, GluA1 = 0.93 ± 0.098, GluA4 = 1.014 ± 0.17. Number of samples: 7, 6, 7, 7 respectively; Kruskal–Wallis test, P = 0.472. Surface: Ctr = 1 ± 0; GluA2/3 = 0.89 ± 0.041, GluA1 = 0.92 ± 0.038, GluA4 = 0.89 ± 0.047; number of samples: 6, 6, 6, 6, respectively. Kruskal–Wallis test, P = 0.112; 3 independent experiments, normalized values, mean ± SEM).C, D(C) Representative traces and (D) quantitative analysis of total current density evoked by application of 30 µM AMPA in control and PTX3-treated cultures (Ctr = 1 ± 0.117; PTX3 = 0.983 ± 0.87; Student's t-test; number of neurons: Ctr = 13, PTX3 = 12; 3 independent experiments, data are presented as distribution plus normalized mean ± SEM).EPTX3 does not affect neuronal pentraxins and glypican 4 expression. qRT–PCR analysis of mRNA levels for NP1, NP2, and glypican 4 (Gpc4) performed in pure neuronal cultures treated with PTX3 (1 µg/ml for 48 h). qPCR fold change normalized on control: Ctr = 1.000 ± 0.000; NP1 = 1.174 ± 0.16; NP2 = 1.041 ± 0.182; Gpc4 = 0.980 ± 0.062. Wilcoxon test: Ctr vs. NP1: P = 0.312, Ctr vs. NP2: P = 0.688, Ctr vs. Gpc4: P > 0.999. Graphs show mean ± SEM. At least 5 independent experiments. Source data are available online for this figure.

Fig 3: GluA2 is retained intracellularly and is expressed less at post-synaptic sites in the granule cell layer of Tg(PG14) mice.(A) HeLa cells were co-transfected with plasmids encoding WT or PG14 PrP-EGFP fusion protein, and the AMPAR subunit GluA2. After 48h cells were fixed, permeabilized, stained with anti-GluA2 (red), and reacted with Hoechst 33258 (blue) to stain the nuclei. (B) The fluorescent density of GluA2 on the cell surface was measured and corrected for background (Corrected IntDen). Each bar indicates the mean ± SEM of 12 WT and 14 PG14 PrP-transfected cells from three independent experiments; *p<0.01 by Student’s t-test. (C) HeLa cells were co-transfected with WT or PG14 PrP and EGFP-tagged GluA4. After 48 h cells were fixed, permeabilized, stained with anti-PrP monoclonal antibody 98A3 (green), and reacted with Hoechst 33258 (blue) to stain the nuclei. (D) The fluorescent density of GluA4 on the cell surface was measured and background corrected. Each bar indicates the mean ± SEM of 6 WT and 6 PG14 PrP-transfected cells. (E) CGNs from WT and PG14 mice were fixed, permeabilized and immunostained with mouse monoclonal anti-GluA2 (green) and chicken polyclonal anti-MAP2 (red). Cells were reacted with Hoechst 33258 (blue) to stain the nuclei, and analyzed by confocal microscopy using sequential Z-stack acquisition mode and 3D reconstruction. The GluA2 signal on dendrites (F) and cell bodies (G) was analyzed by NIH ImageJ software. Data are the mean ± SEM of 18 WT cells (28 dendrites) and 18 PG14 cells (27 dendrites) from three independent experiments. (H-K) Brain sections of WT and PG14 mice were stained with anti-GluA2 (H) or GluA4 (J) (red) and anti-VGLUT1 (green) antibodies. The GluA2 (I) and GluA4 (K) in each cerebellar glomerulus were measured and expressed as the ratio between the area of the AMPA receptor subunit and VGLUT1 signals. The dotted line identifies a single glomerulus used for quantification. Signal outside the ROI has been cleared with ImageJ for clarity. Bars indicate the mean ± SEM of three mice per group (1098–1392 glomeruli). *p < 0.05 by Student’s t-test. Scale bars 20 µm in A, C and E; and 125 µm in H and J.

Fig 4: Mutant PrP interacts with the GluA2 AMPAR subunit impairing its membrane delivery.(A) Hippocampal protein extracts (500 µg) from WT, FFI and CJD mice were incubated with uncoated magnetic beads (IP: no Ab) or magnetic beads coated with anti-PrP monoclonal antibody 94B4 (IP: PrP). The immunoprecipitated proteins were analyzed by western blot with anti-GluA1 or anti-GluA2 antibody or anti-PrP polyclonal antibody P45-66. The input is 20 µg of total proteins. Lanes 4 and 8 were left empty on purpose. This experiment is representative of three similar ones. (B) HeLa cells were co-transfected with plasmids encoding WT, FFI or CJD PrP-EGFP fusion protein, and the AMPAR subunit GluA2. After 48h, cells were fixed, permeabilized, stained with anti-GluA2 (red) antibody, and reacted with Hoechst 33258 (blue) to stain the nuclei. Scale bar 10 µm. (C, D) The fluorescent density of PrP and GluA2 on the cell surface was measured and background corrected (Corrected IntDen). Each bar indicates the mean ± SEM of 12–17 tranfected cells from three independent experiments. **p < 0.01, ****p < 0.0001 by one-way ANOVA, Tukey’s post hoc test.

Fig 5: PrP and GluA2 co-immunoprecipitate from cerebellar extracts.(A) Cerebellar protein extracts (500 μg) from Tg(WT), Tg(PG14) and PrP knockout (KO) mice were incubated with uncoated magnetic beads (IP: no Ab) or magnetic beads coated with anti-PrP monoclonal antibody 94B4 (IP: PrP). The immunoprecipitated proteins were analyzed by western blot with anti-GluA2 or anti-GluA4 antibody or anti-PrP polyclonal antibody P45-66. The input is 20 μg of total proteins. (B, C) The amounts of immunoprecipitated GluA2 (B) and GluA4 (C) were quantified by densitometry of western blots like in (A) and normalized on the amount of immunoprecipitated PrP. Data are the mean ± SEM of 3–4 replicates from three independent experiments; **p < 0.01 by Student’s t-test.