Fig. 6: NAD⁺ supplement abrogates Aβ pathological progress and decreases neuroinflammation depending on ATF4-mediated UPR^mt in 5xFAD mice.

a Schematic of NMN injection and behavioral analysis in AAV-atf4 Knowdown mice. b–d The behavioral tests analysis of 6-month-old AAV-atf4 Knowdown mice. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test for (b, c), while two-way ANOVA was utilized to assess memory in the MWM test (d, n = 7 mice per group). e Representative immunostained images and quantification of the diameter and number of both 4G8+ (red) and thioflavin S+ (ThS+, white) amyloid plaques, stained with Aβ-positive microglia (IBA1, green) in the hippocampi of AAV-free AD (NMN) and AAV-atf4 AD (NMN) mice. Scale bar, 10 μm (n = 7 mice; two-sided unpaired t-test). f Representative images showing microglial cells (red) and astrocytes (green) engulfing ThS-immunostained amyloid plaques. Quantification was performed in two independent experiments in at least 10 ROIs. Scale bar, 10 μm. g, h Corresponding quantification of microglial cells and astrocytes intensity in (f) (n = 5 mice; two-sided unpaired t-test). i Effects of NMN on the expression levels of APP and its indicated metabolites, MSR analyses of hippocampal brain tissues samples of 5xFAD mice injected either AAV-free or AAV-atf4, following NMN treatment (n = 3–6 animals per group). j–l Corresponding quantification of western blot data in (i). Data were analyzed by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. m Soluble and insoluble Aβ_1–40 and Aβ levels in 5xFAD (NMN) mice injected with AAV-free or AAV-atf4 hippocampal tissues. (n = 7 mice; two-sided unpaired t-test). All experiments were performed independently with at least three biological replicates with similar results. Data are shown as mean ± s.e.m. The p-values are indicated on the graphs. ns not significant.