Fig 2: Mn exposure promoted amyloidogenic APP processing in 3×Tg-AD mouse brains. (A–D) The expression levels of BACE1, ADAM10, and γ-secretase PS1 in the brain of Alzheimer’s disease (AD) were examined by Western blot. The protein levels of BACE1 and PS1 were significantly increased (B,D), whereas ADAM10 were markedly decreased (C) in the cortex and hippocampus of Mn-exposure mice. (E–G) Western blot analysis of the expression of intracellular APP cleavage fragments, including C99 and C83, in the brain of AD mice treated with or without Mn. Mn exposure significantly increased the levels of C99 both in the cortex and hippocampus, and reduced the levels of C83 in hippocampus. β-actin served as the internal control. n = 5 mice for each group, *p < 0.05, **p < 0.01, compared with the control group, t-test.

Fig 3: Aβ in tau extracts leads to a redistribution of nondense core plaque Aβ on ipsilateral hemisphere.A, immunoblot of control (C) and brain A/B extract probed for Aβ with D54D2 antibody and compared with different amounts of synthetic biotinylated-Aβo. Samples were boiled for 5 min at 95 °C with 10% BME. B, quantification of the mean percent area occupied by D54D2-positive and ThioS-positive dense core plaques in the hippocampus of control and AD brain–injected mice was measured by drawing ROIs around the brain region and thresholding images. Representative images can be found in C and Fig. S3A. Statistics: Ordinary two-way ANOVA (for D54D2—interaction: F(5, 43) = 5.939, p = 0.0003; row factor: F(5, 43) = 12.25, p < 0.0001; column factor: F(1, 43) = 11.62, p = 0.0014; for ThioS—interaction: F(5, 43) = 0.4724, p = 0.7947; row factor: F(5, 43) = 5.546, p = 0.0005; column factor: F(1, 43) = 0.09148, p = 0.7638) with Sidak's multiple comparisons test comparing ipsilateral and contralateral hemisphere within each group shown in black and Sidak's multiple comparisons test comparing ipsilateral hemispheres between control and AD-injected mice shown in gray. N represents individual animals. N(APP-C) = 4, N(APP-AD) = 5, N(hT-C) = 4, N(hT-AD) = 3, N(DKI-C) = 4, and N(DKI-AD) = 7. ∗p < 0.05, ∗∗∗∗p < 0.0001. C, immunofluorescent staining with D54D2 (magenta) antibody for amyloid-β and thioflavin S (green) for dense-core amyloid-β plaques of APP and DKI mice injected with AD brain tau extracts (the scale bar represents 1 mm). D, representative images of D54D2 (magenta) and ThioS (green) staining taken with a 20× objective (the scale bar represents 50 μm). Schematic on the left indicates the location of images in the brain. E, quantification of D54D2 staining seen in D. For quantification of ThioS staining, see Fig. S3B. Statistics: Ordinary two-way ANOVA (for region 1—interaction: F(1, 13) = 2.449, p = 0.1416; row factor: F(1, 13) = 3.066, p = 0.1035; column factor: F(1, 13) = 3.131, p = 0.1002; For region 2—interaction: F(1, 14) = 2.309, p = 0.1509; row factor: F(1, 14) = 5.332, p = 0.0367; column factor: F(1, 14) = 2.655, p = 0.1255; region 3—interaction: F(1, 14) = 2.701, p = 0.1225; row factor: F(1, 14) = 3.560, p = 0.0801; column factor: F(1, 14) = 2.609, p = 0.1285; region 4—interaction: F(1, 14) = 0.005899, p = 0.9399; row factor: F(1, 14) = 0.8148, p = 0.3820; column factor: F(1, 14) = 1.500, p = 0.2410; region 5—interaction: F(1, 14) = 0.5541, p = 0.4690; row factor: F(1, 14) = 0.5692, p = 0.4631; column factor: F(1, 14) = 0.0001762, p = 0.9896; region 6—interaction: F(1, 14) = 7.892, p = 0.0139; row factor: F(1, 14) = 8.591, p = 0.0109; column factor: F(1, 14) = 8.843, p = 0.0100; region 7—interaction: F(1, 14) = 1.918, p = 0.1877; row factor: F(1, 14) = 4.515, p = 0.0519; column factor: F(1, 14) = 2.662, p = 0.1251; region 8—interaction: F(1, 14) = 0.3087, p = 0.5879; row factor: F(1, 14) = 1.470, p = 0.2470; column factor: F(1, 14) = 0.02451, p = 0.8780) with Sidak's multiple comparisons test comparing ipsilateral and contralateral hemisphere within each injection and ipsilateral results across injections. N represents individual animals. N(APP-C) = 4, N(APP-AD) = 4 and 5. ∗p < 0.05, ∗∗p < 0.01. AD, Alzheimer's disease; APP, amyloid precursor protein; DKI, double KI.

Fig 4: BCAA restriction delays the onset of cognitive deficit in APP/PS1 mice. 11-month-old WT or APP/PS1 mice without cognitive impairment were placed on a control diet or BCAA-restricted (50%) diet, iso-caloric and iso-nitrogenous, for two months. (A) Plasma BCAA levels at baseline. (B) BCAA restriction does not affect body weight or (C) Food intake. (D) Blood glucose before and after treatment. (E) Schematic of Y-maze behavioral test. (F) Spontaneous alternation (%) before and after treatment. (G) Total distance traveled, (H) Freezing time, (I) Number of arm entries, and (J) Mean speed during Y-maze test. WT control or BCAA restriction group (n = 10/group); APP/PS1 control or BCAA restriction group (n = 8/group). * p < 0.05.

Fig 5: Immunodepleting Aβ from tau extracts ameliorates Aβ redistribution.A, immunoblots of tau extracts that had been incubated with D54D2 antibody conjugated to Protein G magnetic beads to remove Aβ from tau extracts. Blots shown were probed for Aβ (D54D2) to assess the removal of Aβ from the extracts. Bands shown were run on the same blot but not next to each other, and vertical black lines indicate splicing of the blot. U = untreated samples, D = D54D2-incubated samples. B, mean number of somatic inclusions on the ipsilateral hemisphere of animals injected with control (C) or AD brain tau extracts, or with control or AD brain tau extracts that had been cleared of Aβ by incubating samples with D54D2 antibody conjugated to Protein G beads (C-D and AD-D samples). Statistics: Kruskal–Wallis test (approximate p value = 0.0176, Kruskal–Wallis statistic = 16.97) with Dunn's multiple comparisons test. N represents individual animals. N(WT-C) = 4, N(WT-C-D) = 4, N(WT-AD) = 8, N(WT-AD-D) = 7, N(APP-C) = 2, N(APP-C-D) = 1, N(APP-AD) = 8, and N(APP-AD-D) = 10. ∗p < 0.05. C, schematics of mean somatic inclusion burden of tau extract (extracted from AD brains, with or without Aβ removal)–injected animals dependent on mouse genotype. Brain regions not analyzed are depicted in gray. Ipsilateral hemisphere is on the right. D, area occupied by neuritic inclusions in four brain regions of animals injected with control or AD brain–derived tau extracts with and without Aβ removal. Statistics: Ordinary two-way ANOVA (for dorsal hippocampus—interaction: F(7, 68) = 1.680, p = 0.1287; row factor: F(7, 68) = 11.67, p < 0.0001; column factor: F(1, 68) = 1.790, p = 0.1864; for ventral hippocampus—interaction: F(7, 70) = 0.06833, p = 0.9995; row factor: F(7, 70) = 3.114, p = 0.0064; column factor: F(1, 70) = 0.07003, p = 0.7921; for fimbria—interaction: F(7, 61) = 0.7205, p = 0.6550; row factor: F(7, 61) = 1.275, p = 0.2777; column factor: F(1, 61) = 2.317, p = 0.1331; for corpus callosum—interaction: F(7, 67) = 0.2680, p = 0.9573; row factor: F(7, 67) = 1.825, p = 0.0967; column factor: F(1, 67) = 0.5514, p = 0.4603) with Sidak's multiple comparisons test. In gray: comparing ipsilateral and contralateral hemisphere for the same genotype and injected extract. In black: comparing ipsilateral values of different tau extract–injected groups. N(WT-C) = 4, N(WT-C-D) = 2, N(WT-AD) = 8, N(WT-AD-D) = 6 and 7, N(APP-C) = 2, N(APP-C-D) = 1, N(APP-AD) = 8, and N(APP-AD-D) = 10. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.0001. E, representative images of the ipsilateral hippocampus with immunofluorescent staining of D54D2 (magenta) antibody for Aβ and ThioS (green) for dense-core amyloid plaques in APP mice injected with AD brain extracts with and without Aβ removal (the scale bars represent 250 μm). F, quantification of the percent area occupied by D54D2 (top) and ThioS (bottom) in the hippocampus of APP mice injected with AD brain extracts with and without Aβ removal. Statistics: Ordinary two-way ANOVA (for D54D2—interaction: F(1, 29) = 0.9156, p = 0.3465; row factor: F(1, 29) = 5.346, p = 0.0281; column factor: F(1, 29) = 10.96, p = 0.0025; for ThioS—interaction: F(1, 32) = 0.2028, p = 0.6555; row factor: F(1, 32) = 0.08193, p = 0.7765; column factor: F(1, 32) = 0.02327, p = 0.8797) with Sidak's multiple comparisons test comparing ipsilateral and contralateral hemisphere within each injection. N represents individual animals. N(APP-AD) = 8, N(APP-AD-D) = 8 to 10. ∗p < 0.05. AD, Alzheimer's disease; APP, amyloid precursor protein; DKI, double KI.