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. 2023 Feb 26;19(8):2318–2337. doi: 10.1080/15548627.2023.2181614

Figure 5.

Figure 5.

Phosphorylated MAPT disrupts V-ATPase activity by interacting with ATP6V1B2. (A) Images of primary hippocampal neurons (DIV 7–9) that were transduced with AAV-GFP or AAV-MAPTP301L::GFP and further treated with 5 μM Aβ for 24 h (left). Cells were co-treated with DQ-red-BSA and Cascade Blue-dextran. Proteolysis activity was analyzed by measuring DQ-red-BSA:Cascade Blue-dextran fluorescence ratio (n = 3, cells = 22–27 in total), two-way ANOVA (right). (B) Representative images showing lysosomal activity in mouse hippocampal CA3 regions. Five-month-old tTA−/−MAPT± and tTA±MAPT± mice were i.c.v. injected with DQ-red-BSA and Cascade Blue-dextran (shown in Fig. S3B). Scale bar: 100 μm (left). DQ-BSA intensity was normalized to the Cascade Blue signal; tTA−/−MAPT± (n = 3); tTA±MAPT± (n = 3), two-tailed unpaired t-test (right). (C) Co-IP analysis of homogenates prepared from HEK293T cells co-expressing EGFP-ATP6V1B2 and either HA-MAPT WT or MAPTS396A,S404A mutant (shown in Fig. S5C) (upper). The relative levels of MAPT-bound to ATP6V1B2 were measured (n = 3), two-tailed unpaired t-test (lower). (D) Microsomes purified from mouse brains were incubated for 40 min with 1 μM Baf.A1, 3 μM BSA, 2 μM His-tagged MAPT WT, or MAPTS396A,S404A mutant protein purified from HEK293T cells, and the free phosphate levels were measured (n= 3), two-way ANOVA. (E) HT22 cells stably expressing pHLARE were transfected with either empty vector (-), HA-tagged MAPT WT, or HA-MAPTS396A,S404A mutant for 24 h. The pH was measured by calibrating sfGFP:mCherry fluorescence ratio (n = 3, each dot presents the average pH of cells > 300 for each trial), one-way ANOVA. (F) Domain structure of ATP6V1B2 WT and deletion (Δ) mutants; N, N-terminus; NBD, nucleotide-binding-domain; C, C-terminus and their binding ability to MAPT. (G) HEK293T cells were co-transfected with indicated EGFP-ATP6V1B2 constructs and HA-MAPT WT for 24 h. Cell homogenates were subjected to co-IP using anti-HA antibody. (H) Images of HT22 cells that were co-transfected with empty vector, or HA-MAPT and either with EGFP, EGFP-ATP6V1B2 WT or Δ120-172 constructs for 24 h. Cells were co-treated with DQ-red-BSA and Cascade Blue-dextran for 6 h. (A, H) Scale bar: 10 μm.