fig6

Figure 6. VAPB P56S mutation induces activation of IRE1-XBP1/p38 MAPK/JNK pathway, hyperphosphorylation of tau, and upregulation of total and phosphorylated p53. (A and B) Western blotting of IRE1-XBP1/p38 MAPK/JNK pathway proteins in the cortex of WT and P56S KI mice at 12 months of age (n = 4); (C-F) Immunostaining of p-IRE1α (Ser724) (C) or spliced XBP1 (E) in the CSMNs of WT and P56S KI mice at 12 months of age (n = 5); (G) The cell viability of cultured WT and P56S KI cortical neurons was measured by MTT assay on DIV7, 14, 21, and 28 (n = 30 at each time point); (H) DIV14 WT and P56S KI cortical neurons were treated with 0 or 1 m JNK inhibitor, SP600125, for 14 days and were assayed for cell viability (n = 28). Scale bar: 20 μm in (C and E). Unpaired t test: ^*P = 0.0185 [p-IRE1α (Ser724)], not significant (ns) P = 0.8588 (IRE1α), ^* P = 0.0173 (spliced XBP1), ^**P = 0.0020 [p-p38 MAPK (Thr180/Tyr182)], ns P = 0.9443 (p38MAPK), ^****P < 0.0001 [p-JNK (Thr183/Tyr185)], ns P = 0.9267 (JNK), ^***P = 0.0003 [p-tau (Ser202/Thr205)], ^**** P < 0.0001 [p-tau (Thr212/Ser214)], ^****P < 0.0001 [p-tau (Thr231)], ns P = 0.8587 (tau), ^**P = 0.0011 [p-p53 (Ser15)], and ^****P < 0.0001 (p53) in (B); ^****P < 0.0001 in (D); ^***P = 0.0002 in (F). Two-way ANOVA: ns P = 0.747006536694 (DIV7), ns P = 0.9999 (DIV14), ^****P < 0.0001 (DIV7, DIV21, and DIV28) in (G). One-way ANOVA: ^****P < 0.0001 (P56S KI vs. WT), ns P = 0.9898 (WT SP60025 vs. WT), ^*P = 0.0308 (P56S KI SP60025 vs. P56S KI), and ^*P = 0.0214190311 (P56S KI SP60025 vs. WT SP60025) in (H). VAPB: Vesicle-associated membrane protein-associated protein B; IRE1: inositol-requiring enzyme 1; XBP1: X-box binding protein 1; MAPK: mitogen-activated protein kinase; JNK: c-Jun N-terminal kinase; WT: wild-type; KI: knock-in; MTT: 3- (4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide; ANOVA: analysis of variance; Ser: serine.