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. 2022 Aug 2;12(8):e1003. doi: 10.1002/ctm2.1003

FIGURE 2.

FIGURE 2

Knockdown nuclear factor‐erythroid 2–related factor 2 (NRF2) induces learning and memory deficits as seen in P301S mice. (A) Experimental procedures of virus injection and behavioural tests. AAV‐P301S‐EGFP or AAV‐VEC, AAV‐sgNRF2‐Cas9 or AAV‐Cas9 were stereotaxically injected into the hippocampal CA3 region of 2‐month‐old C57BL/6J mice. The capacity of learning and memory was detected 4 weeks later. (B) Efficiency of virus infection at CA3 was confirmed by GFP fluorescence. Bar, 500 μm. (C and F) The expression of P301s or NRF2 was confirmed by Western blotting. One‐way analysis of variance (ANOVA), F(3, 8) = 16.41, p =.0032; n = 3. (D and E) Overexpressing P301S increased malondialdehyde (MDA) concentration while reduced superoxide dismutase (SOD) activity in CA3 of C57BL/6J mice. One‐way ANOVA, [MDA] F(3, 8) = 23.13, p =.0081; [SOD] F(3, 8) = 19.53, p =.0043; n = 3. (G and H) Knockdown NRF2 decreased mice's staying time in centre zone of the box of open field test (OFT). One‐way ANOVA, F(3, 36) = 4.127, p = .0130; n = 10. (I and J) Knockdown NRF2 increased mice's staying time in frame of elevated plus maze (EPM) test. One‐way ANOVA, F(3, 36) = 19.23, p < .0001; n = 10. (K and L) Knockdown NRF2 impaired novel object recognition (NOR) as seen by overexpressing P301S. Cartoons show paradigms of NOR test. One‐way ANOVA, F(3, 37) = 3.479, p = .0254; n = 9–11 mice in each group. (M and N) Knockdown NRF2 induced spatial learning deficit as seen in P301S overexpressing measured by Morris water maze (MWM) test. Two‐way ANOVA × days, F(3, 180) = 4.116, p = .0075; n = 9–10 mice in each group. Cartoons show paradigms of MWM test. (O‐R) Knockdown NRF2, as seen by overexpressing P301S, impaired spatial memory shown by the decreased platform crossing times (O), decreased duration in the target quadrant (P) and an increased latency to reach the platform location (Q) in water maze detected at day 7 after removing the platform; the swimming speed was not changed by knockdown NRF2 or overexpressing P301S (R). One‐way ANOVA, [platform crossing times] F(3, 36) = 5.268, p = .0041; [duration] F(3, 36) = 11.76, p < .001. [Latency] F(3, 36) = 5.055, p = .005; n = 10 mice in each group. (S and T) Knockdown NRF2 impaired contexture memory, as seen in P301S overexpression, measured by fear conditioning test (FCT). One‐way ANOVA, F(3, 36) = 4.967, p = .0055; n = 10 mice in each group. * p < .05, ** p < .01 versus VEC; # p < .05, ## p < .01 versus Cas9. Data were presented as mean ± SEM.