Fig 2: P2X4 regulates cathepsin B-dependent ApoE degradation. A, B Comparison of treatment with E64, a pharmacological inhibitor of the cysteine proteases, on ApoE expression in BMDM culture of WT and P2X4 KO mice. A Representative Western blot of ApoE in the supernatant of WT and P2X4 KO BMDM after incubation with 10 µM E64 overnight. B Quantitative analysis of Western blots shows that E64 induced a strong increase of ApoE in the supernatant of WT but not in P2X4 KO BMDM. N = 5 independent experiments, **p < 0.01, One sample t test compared to theoretical value of 1. C, D Comparison of treatment Z-Phe-Ala-FMK, a CatB inhibitor, on ApoE expression in BMDM culture of WT and P2X4 mice. C Representative Western blot of ApoE in the supernatant of WT and P2X4 KO BMDM after incubation with 20 µM Z-Phe-Ala-FMK overnight. D Quantitative analysis shows that inhibition of CatB with Z-Phe-Ala-FMK induces a strong increase of ApoE in the supernatant of WT but not in P2X4 KO BMDM. N = 6 experiments, **p < 0.01, One sample t test compared to theoretical value of 1. E, F Co-localization in BMDM of P2X4, ApoE and CatB in CD68-positive compartments. E Representative picture of CD68 (green), P2X4 (red) and CatB (white) immunostaining in BMDM cells. Scale bar 5 µm. F Representative immunostaining of ApoE (green), P2X4 (red) and CatB (white), DAPI (blue) in BMDM cells. Images are representative of N = 3 experiments. Scale bar 5 µm. G–I P2X4 regulates CatB activity in BMDM. G CatB activity was measured using the cell-permeable fluorogenic CatB substrate Z-RR-AMC. After incubation with 100 µM Z-RR-AMC, fluorescence was read 1 h and 2 h later. A significant increase of the signal is observed in WT macrophages between 1 and 2 h, whereas the activity in KO cells remained unchanged. N = 8 experiments, **p < 0.01, One sample t test compared to theoretical value of 1 WT(1 h) vs KO (1 h) and WT (1 h) vs WT(2 h), $ p < 0.05 Kruskal–Wallis test WT (2 h) vs KO (2 h); KO (1 h) vs KO (2 h) is non-significant. H Representative microscopic image of cellular CatB activity in WT and P2X4 KO BMDM using the Magic red cathepsin B kit. A strong signal is observed in WT BMDM as compared to P2X4 KO cells. I Quantitative analysis of the magic Red fluorescence using ImageJ. *p < 0.05, N = 3 independent cultures, unpaired t test. Scale bar 30 µm

Fig 3: Interaction between P2X4 and ApoE is recapitulated in recombinant system. A Representative immunofluorescence of ApoE (green), P2X4 (red), and DAPI (blue) in ApoE or ApoE + P2X4 co-transfected COS-7 cells. Both ApoE and P2X4 co-localize in intracellular compartments. Images are representative of N = 3 independent experiments. Scale bar 10 µm. B, C Comparison of ApoE expression upon co-transfection with P2X4. COS-7 cells were transfected with ApoE alone or in combination with P2X4. B Expression of ApoE was analyzed by Western blot in both cell culture supernatants and cell lysates. C Quantitative analysis shows that in the presence of P2X4, amounts of ApoE is reduced in both culture supernatant (Sup) and cell lysates (Lys). N = 3 independent experiments, **p < 0.01, One sample t test compared to theoretical value of 1. D, E Comparison of ApoE expression upon co-transfection with P2X4 or P2X2. D Expression of ApoE was analyzed by Western blot in both cell culture supernatants and cell lysates. E Quantitative analysis of ApoE in supernatant shows that co-expression with P2X4 reduces the expression of ApoE, whereas that of P2X2 has no effect. N = 6 independent experiments, *p < 0.05, One sample Wilcoxon compared to theoretical value of 1 attributed to ApoE + P2X4. F, G P2X4 activity is not necessary to reduce ApoE levels. F Expression of ApoE was analyzed by Western blot in cell culture supernatants of cells transfected with ApoE alone or in combination of either P2X4 or P2X4-K69A, an ATP-binding site dead mutant. G Quantitative analysis shows that both and P2X4 P2X4-K69A significantly reduces the ApoE levels to the same extend. N = 8 independent experiments, **p < 0.01, one sample t-test compared to theoretical value of 1 attributed to ApoE + P2X4

Fig 4: P2X4 interacts with ApoE in BMDM endo-lysosomal compartments and reduces its amount compared to P2X4-deficient cells. A, B Co-immunoprecipitation of P2X4 and ApoE. BMDM membrane extracts from WT and KO mice were immunoprecipitated (IP) with anti-P2X4 (A), or ApoE antibodies (B). Immunoprecipitated proteins were separated by electrophoresis and immunoblotted with either anti-ApoE (top row) or anti-P2X4 (bottom row) antibodies. C Representative immunofluorescence image showing the co-localization of the lysosomal marker CD68 (green), P2X4 (red) and ApoE (purple) in BMDM cells. Scale bar 5 µm. D Representative immunofluorescence image showing a similar localization of ApoE in CD68 + lysosomes in WT and P2X4KO BMDM cells. Scale bar 5 µm. All immunocytochemistry experiments were replicated at least three times. E Representative Western blot of ApoE in BMDM culture supernatants (Sup) or cell lysates (Lys) from WT and KO mice. F Quantitative Western blot analysis presented in E. A significant increase of ApoE is observed in both KO cultures supernatants and in cell lysates. N = 6 independent experiments, *p < 0.05, unpaired t test

Fig 5: Increased ApoE in microglia from APP/PS1xKO mice. A Immunofluorescence of ApoE (blue), Iba1 (green) and P2X4 (red) in APP/PS1 mice cortex. Scale bar 10 µm. B Immunofluorescence of Iba1 (green), P2X4 (Blue) and CD68 (red) in APP/PS1 mice cortex showing the expression of P2X4 in microglial lysosomes. Inset: higher magnification fields. All images are representative of N ≥ 6 experiments, n ≥ 6 mice. Scale bars 50 µm and 5 µm for high magnification. C–E Analysis of ApoE expression in FACS-sorted microglia from APP/PS1 and APP/PS1xKO mice. C Microglia were sorted based on CD11b-PE positive selection. D Representative Western blot of ApoE from APP/PS1 and APP/PS1xKO FACS-sorted cortical microglia. E Quantitative analysis of signals presented in C shows an increase in ApoE in APP/PS1xKO mice relative to APP/PS1 mice. N = 2 independent experiments, n = 2 mice per group