Fig. 1

CircAPP is upregulated in microglia, and knockdown of circAPP in microglia improves cognitive function in APP/PS1 mice and modulates the microglial polarization in vitro. (A) A volcano plot showed dysregulated circRNAs in three hippocampal samples from APP/PS1 and WT mice identified via circRNA microarray, n = 3 samples/group. (B) Sanger sequencing of circAPP. (C) RT-PCR assay was performed to detect circAPP expression in the heart, liver, spleen, lung, kidney, cortex and hippocampus, n = 4. (D ~ F) RT-PCR assay was used to detect the expression of circAPP in 3- (D), 6- (E) and 10- (F) month-old APP/PS1 and age-matched WT mice, n = 4. (G) RNase R resistance of circAPP was assessed by RNase R treatment assay, n = 3. (H) The expression of circAPP in Aβ-treated primary neurons and glial cells was measured using RT-PCR assay, n = 3. (I) The expression of circAPP in Aβ-treated N2a, BV-2 and C8-D1A cells was detected using RT-PCR assay, n = 3 ~ 4. (J) The efficiency of infection of AAV2/9 containing a control shRNA with EGFP driven by microglia-specific promoters was evaluated using IF assay. AAVs (green) colocalized with a microglial marker, Iba-1 (red), in the hippocampus as indicated by white arrows. (K) RT-PCR assay was performed to measure circAPP expression in the hippocampus of mice after infection with shcircAPP AAVs for 6 weeks, n = 4. (L ~ N) The effect of circAPP knockdown on the cognitive function of APP/PS1 mice was evaluated using MWM test (L), passive avoidance test (M) and novel object recognition task (N), n = 8 ~ 10 mice. (O) The effect of circAPP knockdown on Iba-1 expression in the hippocampus of APP/PS1 mice was assessed using Western blot assay, n = 4. (P) CircAPP expression was detected using RT-PCR assay after BV-2 cells were transfected with shcircAPP LVs, n = 3. (Q ~ V) The effects of circAPP knockdown on the expression of Iba-1, TNF-α, pro- and cleaved IL-1β, CD16, Arg1 and IL-10 in Aβ-treated BV-2 cells were evaluated using Western blot assay, n = 3 ~ 4. All data in the figure are presented as mean ± SEM. Student’s t test (D ~ I, K ~ P) and one-way ANOVA (R ~ V) were used to assess statistically significant differences. *P < 0.05, **P < 0.01