Results: 5

1.
Scheme 1

Scheme 1. From: Regulation of forkhead transcription factor FoxO3a contributes to calorie restriction-induced prevention of Alzheimer's disease-type amyloid neuropathology and spatial memory deterioration.

Scheme illustrates the potential relationship for CR, FoxO3a inactivation/SIRT1 activation and promotion of non-amyloidogenic processing of APP.

Weiping Qin, et al. Ann N Y Acad Sci. ;1147:335-347.
2.
Fig. 3

Fig. 3. SIRT1-mediated FoxO3a deacetylation is involved in FoxO3a modulation of Aβ generation. From: Regulation of forkhead transcription factor FoxO3a contributes to calorie restriction-induced prevention of Alzheimer's disease-type amyloid neuropathology and spatial memory deterioration.

(A) Acetylation of FoxO3a decreases in the brain of CR treated Tg2576 transgenic mice. Endogenous FoxO3a was immunoprecipitated by anit-FKHRL-1 antibody followed by western blot using acetyl- lysine antibody. Total levels of FoxO3a were detected by western blot with anit-FKHRL-1 antibody. (B) SIRT1 deacetylates FoxO3a in cultured Tg2576 neurons. Tg2576 neurons were infected with SIRT1 adenovirus (MOI=10) for 48h. The acetylation of immunoprecipitated FoxO3a and total levels of FoxO3a were analyzed by western blot as described in (A). (C) Tg2576 neurons were infected with adeno-GFP or CA FoxO3a virus in combination with Ad-lacZ or Ad-WT SIRT1 viral infection. The resulting conditioned medium and cell lysates 48 hr post-transfection/infection were assessed for Aβ1-40 and Aβ1-42 concentrations. (C) Inset. Western blot analysis of SIRT1 and FoxO3a expressions. In (A) values represent means ± SEM, n = 4–5 per group. In (B) and (C), values represent means ± SEM of determinations made in three separate culture preparations; n=3 per culture preparation. In (A-C), *p<0.05 versus control group (2-tailed Student’s t test).

Weiping Qin, et al. Ann N Y Acad Sci. ;1147:335-347.
3.
Fig. 4

Fig. 4. FoxO3a activates mouse ROCK1 promoter, which is inhibited by viral SIRT1 expression. From: Regulation of forkhead transcription factor FoxO3a contributes to calorie restriction-induced prevention of Alzheimer's disease-type amyloid neuropathology and spatial memory deterioration.

(A) Schematic representation of the ROCK1-luc reporter gene construct in which expression of the reporter luciferase gene is under the control of a ~500 bp mouse ROCK1 promoter sequence encompassing −2549 nt to −2017 nt of the ROCK1 promoter region. (B) CHO-APPswe cells were seeded at a density of 1×105 cells/well in 24-well-plates. 18 hours later, cells were transiently transfected with the ROCK-luc reporter construct using Lipofectamine, following the manufacturer’s instructions. Media were changed 5 hours later and then cells were infected with CA FoxO3a or SIRT1, or both FoxO3a and SIRT1 adenoviruses. Control cultures were transfected with the ROCK1-luc reporter gene construct while simultaneously infected with a green fluorescent protein adenovirus (GFP). Luciferase activities were measured 24h after transfection using Dual-Luciferase Reporter Assay System. (C) Viral CA FoxO3a in CHO-APPswe cells cultures results in increased levels of ROCK1 protein expression. Resulting cell lysates were separated by SDS-PAGE and probed with a rabbit polyclonal antibody against ROCK1; β-actin immunoreactivity served as a loading control. Value are expressed as mean ± SEM from two independent studies, n=3 repetitions per study. * p<0.05 (student t-test) vs. GFP infection.

Weiping Qin, et al. Ann N Y Acad Sci. ;1147:335-347.
4.
Fig. 1

Fig. 1. CR activates IR signaling cascades in the brains of Tg2576 mice. From: Regulation of forkhead transcription factor FoxO3a contributes to calorie restriction-induced prevention of Alzheimer's disease-type amyloid neuropathology and spatial memory deterioration.

In this study ~10 month-old Tg2576 transgenic mice were assessed for indexes of IR signaling in the cerebral cortex in response to CR or AL dietary regimens. (A) Total IRβ and (B) Y1162/1163-phosphorylated IRβ protein contents in the cerebral cortex. Total IRβ expression is normalized to actin; IRβ Y1162/1163 phosphorylation is expressed as the ratio of IRβ Y1162/1163 phosphorylated protein relative to IRβ in the same cerebral cortex samples; (A and B) insets, representative western blot analysis of IRβ and Y1162/1163 phosphorylated IRβ proteins. (C) PI3K (p85α) protein content in the cerebral cortex normalized to actin; (c inset), representative PI3K (p85α) western blot analysis. (D) pS473AKT expressed as a ratio of actin; Total AKT expressed as a ratio of actin; (D) insets, representative western blot analysis of pS473AKT and total AKT. (E) Thr32-p-FoxO3a expressed as a ratio of total FoxO3a; (E) inset, representative western blot analysis of Thr32-p-FoxO3a and total FoxO3a protein. In (A–E), values represent means ± SEM, n = 4–5 per group; * P < 0.05 vs. control group (2-tailed Student’s t test). (F) Cellular distribution of neocortical FoxO3a immunoreactivity in the contralateral neocortex used for (A–E) Western blot analysis. The brain section were fixed and immunofluorescence staining for FoxO3a (a) using anit-FKHRL-1 antibody, which is visualized using Texas Red. Nuclei were identified using DAPI staining (blue). Scale bar: 20μM. Data are expressed as mean ± SEM for 3 animals per group (n = 100 cells per animal).

Weiping Qin, et al. Ann N Y Acad Sci. ;1147:335-347.
5.
Fig. 2

Fig. 2. Exogenous triple mutant constitutive-active (CA) FoxO3a expression causally prevents non-amyloidogenic processing of APP coincidental with increased Aβ1-40 and Aβ1-42 contents in the conditioned medium of cortico-hippocampal Tg2576 neurons. From: Regulation of forkhead transcription factor FoxO3a contributes to calorie restriction-induced prevention of Alzheimer's disease-type amyloid neuropathology and spatial memory deterioration.

(A) Tg2576 neuron cultures were infected with adenovirus expressing GFP or CA FoxO3a for 48h. Resulting cell lysates were subjected to western blot analysis of adenovirus mediated-FoxO3a expression and Thr32-p-FoxO3a content using anti-FoxO3a antibody and Thr32-p-FoxO3a antibody, respectively. (B) Subconfluent Tg2576 neurons were cultures on Lab-Tek chamber slides and infected with CA FoxO3a adenovirus for 24 h. The cells were then fixed and immunofluorescence staining for FoxO3a (a) using anit-FKHRL-1 antibody, which is visualized using Texas Red. In (B) nuclei were identified using DAPI staining (blue). Scale bar: 20μM. Data are expressed as mean ± SEM for three separate experiments (n= 100 cells per experiment). (C) Adenovirus-mediated over-expression of CA FoxO3a in Tg2576 neuron cultures lead to increased contents of Aβ1-40 and Aβ1-42 released into the culture media. Aβ contents were assessed by ELISA 48 hr post-infection (10 MOI). (D) Assessment of changes in sAPPa concentration and (E) full-length APP (expressed as % of total sAPP and actin immunoreactivity, respectively) in response to adenoviral CA FoxO3a expression in Tg2576 neurons. Results are expressed as a % of control (adeno-GFP) infection; values represent means ± SEM of determinations made in three separate culture preparations; n=3 per culture preparation. *p<0.05 versus control group.

Weiping Qin, et al. Ann N Y Acad Sci. ;1147:335-347.

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