Fig 2: ApoA-I-related pathway regulates the expression of proteins related to anxiety, cognition and spatial learning function, as well as the MAPK pathway in the hippocampus of the mice. (a,b) Representative Western blotting bands and quantification of the anxiety-related protein GAD1/β-actin ratios. (a,c) Representative Western blotting bands and quantification of the cognition- and spatial learning-related proteins (P-CaMKII)/CaMKII, (P-CREB)/CREB ratios. (d,e) Representative Western blotting bands and quantification of the MAPK pathway protein P-P38/P38, P-ERK/ERK ratios. N = 8 per group. All data are shown in bar diagrams, which reflect the arithmetic mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 3: Synaptic potentiation increase spine (synaptic) translocation of CaMKII. (A–D) Representative western blots and bar graphs demonstrating the distribution of CaMKII and T286 pCaMKIIα/T287 pCaMKIIβ in synaptosomal and shaft-cytosol tissue extracts derived from WT hippocampus. (E) Representative immunoblots illustrating the comparative synaptic (spine) and shaft-cytosol expression of CaMKII and T286/T287 pCaMKIIα/β. (F) Bar graph (T-test) illustrating the synaptic translocation of T286/T287 pCaMKIIα/β post L-Glutamate treatment. (G) Schematic illustration of synaptic translocation of T286/T287 pCaMKIIα/β. (1) NMDAR potentiation; (2) post-synaptic Ca++ transient activates SK2; (3) Ca++ transient facilitates T286/T287 CaMKII phosphorylation, and synaptic translocation; (4) increased SK2 activity facilitates removal of synaptic T286/T287 pCaMKIIα/β; and (5) translocation of CaMKII from dendritic spine to shaft-cytosol. (B,D,F; ***p < 0.001).

Fig 4: Generation of Camk2a and Camk2b Mutant Mice and Their Sleep Phenotypes.A A schematic diagram illustrating the strategy using a pair of sgRNAs to mediate the deletion of exons 5 and 6 in Camk2a with sgRNAs CTGGATCACGAAGACCCCTG and ACCTGAAGGTGAGTAACCCT. B A schematic diagram for the deletion of exons 7 and 8 in Camk2b with sgRNAs TTGCAGTCACCTATGTCACG and TGTGTGAGGGGAAACACCTG. C Western blot analysis of CaMKIIα and CaMKIIβ from the brain lysates of mice with the genotypes of Camk2a−/−, Camk2a+/− and Camk2a+/+ (4 mice for each genotype shown here). Grayscale values of each genotype were analyzed. D Western blot analysis of CaMKIIα and CaMKIIβ from the brain lysates of mice with the genotypes of Camk2b−/−, Camk2b+/− and Camk2b+/+ (4 mice for each genotype shown here). Grayscale values of each genotype were analyzed. E–J Analysis of sleep in Camk2a+/+ (n = 7, black curve and bar), Camk2a+/− (n = 10, blue curve and bar) and Camk2a−/− (n = 9, red curve and bar) mice. Profiles showing wake time of each hour in min/hr. E profiles of NREM sleep (F), or profiles of REM sleep (G). The x axis shows zeitgeber time (ZT) with the white box indicating light phase (or daytime) and black box dark phase (or nighttime). Twenty four hours, daytime and nighttime durations of wake (H), NREM sleep (I) or REM sleep (J) shown in mins. K–P Sleep of Camk2b+/+ (n = 11, black curve and bar), Camk2b+/− (n = 12, blue curve and bar) and Camk2b−/− (n = 16, red curve and bar) mice. Profiles of wake time (K), NREM sleep (L) or REM sleep (M). Twenty four hours, daytime and nighttime durations of wake (N), NREM sleep (O) and REM sleep (P). ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001; mean ± standard error of the mean (mean ± SEM). Two-way ANOVA (E, F, G, K, L, M); One-way ANOVA (C, D, H, I, J, N, O, P).

Fig 5: Dox treatment causes an alteration in the synaptic proteins' level. Representative immunoblot showing (A) p-CaMKII (B) α-stargazin (C) BDNF relative levels normalized to beta-actin. Bars represent mean ± SEM; ∗indicates significant difference between control and Dox-treated mice, ∗p < 0.05, ∗∗p < 0.01 n = 3–4 mice per group; Two tailed Student's t-test.