fig5

Figure 5. VAPB P56S mutation induces elevation of cytosolic Ca²⁺, activation of CaMKII, and an increase in CRMP2 phosphorylation; (A and B) Western blotting of VAPB and IP3R in the total lysate, MAM fraction, and cytosolic fraction prepared from the cortex of WT and P56S KI mice at 12 months of age (n = 3); (C and D) Labeling of Fluo-4 AM in DIV28 WT and P56S KI cortical neurons costained with MAP2 (red) and VAPB (n = 6); (E and F) WB of p-CAMKIIα (Thr286), CAMKIIα, p-CAMKIIβ (Thr287), CAMKIIβ, p-CRMP2 (Thr555), p-CRMP2 (Thr514), and CRMP2 in the cortex of WT and P56S KI mice at 12 months of age (n = 4); (G and H) Immunostaining of p-CRMP2, CTIP2, and NeuN (purple) in the motor cortex layer V of WT and P56S KI mice at 12 months of age (n = 5). Scale bar: 50 μm in (C); 20 μm in (G). Unpaired t test: ^****P < 0.0001 (VAPB in the total lysate), not significant (ns) P = 0.223445 (IP3R in the total lysate), ^****P < 0.0001 (VAPB in the MAM fraction), and ^***P = 0.000279 (IP3R in the MAM fraction) in (B); ^**P = 0.0039 in (D); ^****P < 0.0001 [p-CaMKIIα (Thr286)], ns P = 0.3465 (CaMKIIα), ns P = 0.1726 [p-CaMKIIβ (Thr287)], ns P = 0.8117 (CaMKIIβ), ^****P < 0.0001 [p-CRMP2 (Thr555)], ^**P = 0.0051 [p-CRMP2 (Thr514)], and ns P = 0.7529 (CRMP2) in (F); ^***P = 0.000138 in (H). VAPB: Vesicle-associated membrane protein-associated protein B; CaMKII: alcium/calmodulin-dependent protein kinase type II; CRMP2: collapsin response mediator 2; IP3R: inositol 1,4,5-trisphosphate receptor; MAM: mitochondria-associated membranes; WT: wild-type; KI: knock-in; MAP: Thr: threonine; WB: western blotting; MAP2: microtubule-associated protein 2; CaMKIIα: calcium/calmodulin-dependent protein kinase type II subunit alpha; IP3R: inositol 1,4,5-trisphosphate receptor.