Presentation_1_Nicotinamide adenine dinucleotide supplementation drives gut microbiota variation in Alzheimer’s mouse model.pdf

Alzheimer’s disease (AD) is the most common neurodegenerative disease. Growing evidence suggests an important role for gut dysbiosis and gut microbiota-host interactions in aging and neurodegeneration. Our previous works have demonstrated that supplementation with the nicotinamide adenine dinucleotide (NAD+) precursor, nicotinamide riboside (NR), reduced the brain features of AD, including neuroinflammation, deoxyribonucleic acid (DNA) damage, synaptic dysfunction, and cognitive impairment. However, the impact of NR administration on the intestinal microbiota of AD remains unknown. In this study, we investigated the relationship between gut microbiota and NR treatment in APP/PS1 transgenic (AD) mice. Compared with wild type (WT) mice, the gut microbiota diversity in AD mice was lower and the microbiota composition and enterotype were significantly different. Moreover, there were gender differences in gut microbiome between female and male AD mice. After supplementation with NR for 8 weeks, the decreased diversity and perturbated microbial compositions were normalized in AD mice. This included the species Oscillospira, Butyricicoccus, Desulfovibrio, Bifidobacterium, Olsenella, Adlercreutzia, Bacteroides, Akkermansia, and Lactobacillus. Our results indicate an interplay between NR and host-microbiota in APP/PS1 mice, suggesting that the effect of NR on gut dysbiosis may be an important component in its therapeutic functions in AD.</p

Traditional Chinese Nootropic Medicine <i>Radix Polygalae</i> and Its Active Constituent Onjisaponin B Reduce β-Amyloid Production and Improve Cognitive Impairments

<div><p>Decline of cognitive function is the hallmark of Alzheimer’s disease (AD), regardless of the pathological mechanism. Traditional Chinese medicine has been used to combat cognitive impairments and has been shown to improve learning and memory. <i>Radix Polygalae</i> (RAPO) is a typical and widely used herbal medicine. In this study, we aimed to follow the β-amyloid (Aβ) reduction activity to identify active constituent(s) of RAPO. We found that Onjisaponin B of RAPO functioned as RAPO to suppress Aβ production without direct inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase activities. Our mechanistic study showed that Onjisaponin B promoted the degradation of amyloid precursor protein (APP). Further, oral administration of Onjisaponin B ameliorated Aβ pathology and behavioral defects in APP/PS1 mice. Taken together, our results indicate that Onjisaponin B is effective against AD, providing a new therapeutic agent for further drug discovery.</p></div

Onjisaponin B promotes APP degradation.

<p>(A-B) RAPO, RAPO fractions (1 mg/ml) (A) and Onjisaponin B (10 μM) (B) reduce mature APP levels in HEK293-APPswe cells. (C) Generation of sAPPβ in the presence of 10 μM Onjisaponin B or Tenuifolin in BACE1-assay buffer. (D) Mature APP was accumulated in detergent-soluble membrane fractions treated with 10 μM BSI IV together with Onjisaponin B. (E-F) The proteasome inhibitor MG132 (10 μM) prevents the reduction of mature APP (E) and Aβ generation (F) by Onjisaponin B. (F) The proteasome inhibitor lactacystin (20 μM) partially blocks the Aβ reduction by Onjisaponin B. Data are presented as the mean ± s.e.m. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001. Two-way ANOVA with Bonferroni's multiple comparison test (F).</p

Systemic fractionation identifies RAPO-1-3 as the active fraction of RAPO that reduces Aβ production.

<p>(A) The extraction and fractionation scheme of RAPO. (B) RAPO-1-3 significantly reverses the spatial memory deficit of AD mice. (C) Representative tracks of each group of mice in probe trial test at day 8. (D) The latency to platform in probe trial for each group of mice at day 8. (E) No differences in the swimming distance and velocity among the groups. (F) The time spent by mice in the target quadrant. (G) SDS-soluble and FA-soluble Aβ40 and Aβ42 levels in the mouse hippocampi and cortices were measured by sandwich ELISA and normalized to control. Data are presented as the mean ± s.e.m. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001. Two-way ANOVA with Bonferroni's multiple comparison test (B, F), one-way ANOVA with Bonferroni's multiple comparison test (D, E) and two-tailed <i>t</i>-test (G).</p

RP treatment improves locomotor functions, prolongs lifespan and reduces Aβ levels of AD transgenic <i>Drosophila</i>.

<p>CS, Aβ and APP/BACE transgenic flies were cultured on food containing different concentrations of RP (the triangle symbol indicates concentrations from low to high: 0.2, 0.6 and 2 mg/ml) or Memantine (120 µM). (<b>A</b>) Survival curves of flies treated with either RP or Memantine. The data are presented as the mean ± S.E.M. (<b>B</b>) The climbing ability of flies (right panels) was assessed at day 30 for CS and APP/BACE flies and at day 20 for Aβ flies. The values are the mean ± S.E.M. Each value represents the mean of three experiments. (<b>C</b> and <b>D</b>) Aβ and APP/BACE transgenic flies were cultured on SS, AT, PRP or RP (2 mg/ml). Aβ₄₀ and Aβ₄₂ levels in 500 fly heads were measured by ELISA assay. Mem = Memantine. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs.</i> the control group.</p

SS treatment ameliorates learning and memory impairment in Morris Water Maze and Object recognition test.

<p>(<b>A</b>) MWM test for SS and vehicle-treated APP/PS1 and WT mice. The mean escape latency was given for different test days. (<b>B</b>) The mean percent time in probe trial of MWM on day 7. TQ: Target quadrant; AL: Adjacent left; AR: Adjacent right; OP: Opposite. (<b>C</b>) Representative mice search paths from different groups. (<b>D and E</b>) The latency to target quadrant (<b>D</b>) and the frequency to pass the target position (<b>E</b>) in probe trial are shown. (<b>F and G</b>) The swimming velocity (<b>F</b>) and distance (<b>G</b>) in probe trial are shown. (<b>H and I</b>) Novel object recognition analysis. Preference scores of training phase (<b>H</b>) and Recognition Index of testing phase (<b>I</b>) during a 10-min testing phase are shown, respectively. n = 9–12 for each group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, #<i>P</i><0.05, ##<i>P</i><0.01, ###<i>P</i><0.001.</p

SS treatment improves locomotor functions and prolongs lifespan of AD transgenic <i>Drosophila</i>.

<p>(<b>A, C and E</b>) CS, APP/BACE and Aβ transgenic flies were cultured on food containing different concentrations of SS (the triangle symbol stands for concentrations from low to high: 0.2, 0.6 and 2 mg/ml) or Memantine (120 µM). Survival curves for flies treated with either SS or Memantine. The data are presented as mean ± S.E.M. The right panel shows the mean survival days calculated according to the survival curves. (<b>B, D and F</b>) The climbing ability of CS, APP/BACE and Aβ transgenic flies treated with SS or Memantine at day 30 (for CS and APP/BACE flies) and day 20 (for Aβ flies). Values are mean ± S.E.M. Each value represents the mean of three experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs.</i> Ctrl group. Mem = Memantine.</p

AT and PRP improves locomotor function and prolongs lifespan of AD transgenic <i>Drosophila</i>.

<p>CS and Aβ transgenic flies were cultured on food containing different concentrations of AT (0.2, 0.6 and 2 mg/ml), PRP (0.2, 0.6 and 2 mg/ml) or Memantine (120 µM). (<b>A, C</b>) Survival curves for flies treated with either AT, PRP or Memantine. (<b>B, D</b>) The climbing ability of flies was assessed. The values are the mean ± S.E.M. Each value represents the mean of three experiments. Mem = Memantine. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs.</i> the control group.</p

SS treatment alleviates Aβ levels and amyloid plaque burden, reduces gliosis and neuron loss in APP/PS1 mice.

<p>(<b>A–C</b>) Representative half brain sections of WT mice, vehicle or SS-treated APP/PS1 mice stained with antibody against Aβ (6E10) and double staining of GFAP and 6E10 are shown. Scale bar, 1 mm. (<b>B</b> and <b>C</b>) Quantitative analysis of the number of 6E10-positive amyloid plaques (<b>B</b>) and Aβ covered area (<b>C</b>). n = 5 animals per group. (<b>D</b> and <b>E</b>) ELISA of soluble and insoluble Aβ₄₀ and Aβ₄₂ levels in cortical and hippocampal tissues of APP/PS1 mice. n = 6 for each group. (<b>F</b>, <b>I</b> and <b>J</b>) Representative images of WT mice, vehicle- and SS- treated APP/PS1 mice hippocampus and cortex double immunostaining of GFAP and 6E10 (<b>F</b>), CD11b (<b>I</b>) and NeuN (<b>J</b>). Arrows indicate astrocytes surrounding the amyloid plaques. Scale bar, 200 µm. (<b>H</b>) Coincidence of GFAP and Aβ burden in the brains of SS-treated APP/PS1 mice (red; n = 17) and vehicle-treated APP/PS1 mice (black; n = 17; <i>P</i><0.0001). (<b>G</b>, <b>K</b> and <b>L</b>) The histograms depict the mean GFAP (<b>G</b>), CD11b (<b>K</b>), and NeuN (<b>L</b>) positive area ± S.E.M. in three groups. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

RP reduces the Aβ generation in SK-N-SH-APPsw cells.

<p>Aβ₄₂ (<b>A</b>) and Aβ₄₀ (<b>B</b>) in SK-N-SH-APPsw cell culture medium and cell viability (<b>C</b>) after treatment with SS, AT, PRP, RP for 24 hours, respectively (the triangle symbol stands for concentrations from high to low: 3000, 1000, 300 and 100 µg/ml for SS; 1000, 300, 100 and 30 µg/ml for AT, PRP and RP). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001; DAPT: a γ-secretase inhibitor.</p