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. 2014 Jul 9;34(28):9222–9234. doi: 10.1523/JNEUROSCI.1132-14.2014

Figure 4.

Figure 4.

Overexpression of CAST in JNPL3 mice inhibits cdk5 activation, tau hyperphosphorylation, and Sarkosyl-insoluble pathological tau formation. All the animals used in this figure were 18 months old. A, Female WT (lanes 1, 5), CAST (lanes 2, 6), JNPL3 (lanes 3, 7), and CAST × JNPL3 (lanes 4, 8) cortical extracts (n = 4) were immunoblotted with cdk5 activator protein p35 (C-19), human tau (CP27), CAST (H-300), tau hyperphosphorylation-specific (AT-100, AT8, PHF1), and total tau (Tau-5) antibodies. A–D, CAST inhibits conversion of p35 to p25, and hyperphosphorylation of tau (AT-8, A, E). B–E: 1, WT; 2, CAST; 3, JNPL3; 4, CAST × JNPL3. Representative groupings of the four genotypes from separate sets of mice (#1, #2) are shown. E, Overexpression of CAST decreased the number of AT8-positive neurons in JNPL3 mice. Our data indicate that the number of AT8-positive immunoreactive neurons (G, indicated by arrows) is significantly reduced in CAST × JNPL3 mouse cortices compared with JNPL3 mouse cortices [F, p < 0.05; G (G, 1, JNPL3 (n = 13); 2, CAST × JNPL3 (n = 16)] and pons [H, p < 0.05; I (I, 1, JNPL3; 2, CAST × JNPL3 (n = 9 for each genotype)] of 18-month-old animals (male and female mice). F, H, 1, JNPL3; 2, CAST × JNPL3. Scale bar, 20 μm. J, CAST inhibits formation of Sarkosyl-insoluble tau in JNPL3 mice. Hemi-brains of female mice were fractionated in Sarkosyl-containing buffer, then insoluble and soluble fractions (equal volumes) were immunoblotted with Tau-5, PHF1, AT-8, and AT-100 antibodies. Sarkosyl-insoluble 64 kDa pathological tau is increased in JNPL3 fractions, whereas this increase is inhibited in CAST × JNPL3 mice (see arrows in J). Lanes 1, 5, WT; 2, 6, CAST; 3, 7, JNPL3; 4, 8, CAST × JNPL3 fractions (lanes 1–4, insoluble; lanes 5–8, soluble fractions, n = 3–4 for each genotype). Error bars represent SEM. *p ≤ 0.05 is significant: B, F, H, Student's t test; C–E, one-way ANOVA. K, AT8 staining of nucleus accumbens, zona incerta, and inferior colliculus from WT, CAST, JNPL3, and CAST × JNPL3 mice (18-month-old male and female mice, n = 9 for each genotype) presented at 100× magnification. Expression of CAST in JNPL3 mice appears to reduce AT8 staining in all regions. Scale bar, 20 μm. Measurement of neuronal cell sizes in cortex (L) and pons of brainstem (M) reveal no significant differences in neuronal cell size between JNPL3 and CAST × JNPL3 mice (1, JNPL3; 2, CAST × JNPL3). N, Double immunofluorescence staining of cortices from 18-month-old CAST × JNPL3 mice for human CAST and human tau. Vibratome-sectioned cortices of CAST × JNPL3 mice (n = 8) were double-immunolabeled with CAST 3.1 (human CAST) and CP27 (human tau) antibodies (arrows indicate positive double-labeled neurons). For graph B–E: 1, WT; 2, CAST; 3, JNPL3; 4, CAST × JNPL3.