Combined Liver X Receptor/Peroxisome Proliferator-activated Receptor γ Agonist Treatment Reduces Amyloid β Levels and Improves Behavior in Amyloid Precursor Protein/Presenilin 1 Mice

Abstract

Alzheimer disease (AD) is characterized by the extracellular accumulation of amyloid β (Aβ), which is accompanied by a robust inflammatory response in the brain. Both of these pathogenic processes are regulated by nuclear receptors, including the liver X receptors (LXRs) and peroxisome-proliferator receptor γ (PPARγ). Agonists of LXRs have been demonstrated previously to reduce Aβ levels and improve cognitive deficits in AD mouse models by inducing the transcription and lipidation of apolipoprotein E (apoE). Agonists targeting PPARγ reduce the microglial expression of proinflammatory genes and have also been shown to modulate apoE expression. Here we investigate whether a combination therapy with both LXR and PPARγ agonists results in increased benefits in an AD mouse model. We found that the LXR agonist GW3965 and the PPARγ agonist pioglitazone were individually able to increase the levels of apoE and related genes, decrease the expression of proinflammatory genes, and facilitate Aβ decreases in the hippocampus. Combined treatment with both agonists provoked a further increase in the expression of apoE and a decrease in the soluble and deposited forms of Aβ. The decrease in plaques was associated with increased colocalization between microglia and plaques. In addition, the PPARγ agonist in the combined treatment paradigm was able to counteract the elevation in plasma triglycerides that is a side effect of LXR agonist treatment. These results suggest that combined LXR/PPARγ agonist treatment merits further investigation for the treatment of AD.

Keywords: Alzheimer disease; amyloid β (Aβ); apolipoprotein E (apoE); liver X receptor (LXR); microglia; peroxisome proliferator-activated receptor (PPAR).

FIGURE 1.

Combination therapy increases target gene expression and Aβ degradation and decreases inflammatory markers in vitro. A, cultured primary astrocytes were incubated for 24 h with DMSO (μm), 500 nm GW3965, 100 nm pioglitazone, or both doses combined, and then LXR target genes were quantified by immunoblotting and normalized to actin. Representative blots are shown in the right panel. B, conditioned medium from the same treated primary astrocytes was collected, separated by native PAGE, and immunoblotted for apoE. C, primary microglia were treated for 24 h with GW, pio, or GW + pio, followed by the addition of 2 μg/ml Aβ_1–42 for 18 h. Remaining intracellular Aβ was quantified using ELISA and normalized to total protein. D, cultured primary microglia were incubated for 24 h with the indicated concentrations of drug or DMSO for controls, and then 100 ng/ml LPS was introduced to the medium for 12 h. RNA was extracted, and the expression of proinflammatory genes was examined by qPCR analysis. The dotted line indicates baseline transcript levels in the DMSO-only control. The LPS-treated control was preincubated with DMSO for 24 h and then with 100 ng/ml LPS for 12 h (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the vehicle-treated control; ##, p < 0.01; ###, p < 0.001 compared with the LPS-treated control; Student's t test. a.u., arbitrary units.

FIGURE 2.

Nuclear receptor agonists stimulate transcription of LXR target genes in AD mice. A, 6-month-old transgenic (TG) APP_swe/PSENΔE9 mice were orally gavaged with 50 mg/kg/day GW3965, 80 mg/kg/day pioglitazone, or both for 9 days. Cortical/hippocampal homogenate was immunoblotted for ABCA1, ABCG1, and apoE. Representative blots are shown. Veh, vehicle. B, each sample was normalized to actin, and results are expressed as fold difference compared with vehicle controls. C, equal volumes of whole-brain homogenate from each treatment group were analyzed by native PAGE and immunoblotted for apoE (n = 7–11 animals/group). D and E, 6-month-old transgenic APP_swe/PSENΔE9 or non-transgenic littermate control animals were orally gavaged with the doses described above for 9 days. D, plasma was collected and analyzed for triglyceride content by colorimetric assay (n = 6 animals/group). E, complete livers were removed from each animal and weighed, and liver weight was normalized to total body weight for each animal. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with vehicle-treated control; #, p < 0.05 compared with GW-treated group; Student's t test. a.u., arbitrary units.

FIGURE 3.

Nuclear receptor agonists reduce the proinflammatory environment in APP/PS1 mice. A and B, transcript levels from RNA isolated from whole-brain homogenate (A) and quantification of immunofluorescence in the cortex and hippocampus (B) of the microglia marker Iba1 in 6-month-old APP_swe/PSENΔE9 mice orally gavaged with the indicated drugs for 9 days. Nine hemicoronal sections per animal were immunostained for Iba1, and Iba1 area analysis was performed as described under “Experimental Procedures.” TG, transgenic; Veh, vehicle. C, representative images of Iba1 immunostaining in the cortex. D–G, transcript levels of the proinflammatory markers CD45 (D), IL6 (E), IL1β (F), and TNFα (G) were analyzed by qPCR on RNA isolated from whole-brain homogenate (n = 7–11 animals/group). *, p < 0.05; **, p < 0.01 compared with vehicle-treated APP/PS1 mice; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 compared with non-transgenic littermate control mice. A and D–G, one-way ANOVA with Tukey post-test. B, Student's t test.

FIGURE 4.

Combination therapy significantly reduces the amyloid burden in AD mice. A and B, Aβ was sequentially extracted from whole brain homogenates using diethylamine for soluble Aβ (A) and formic acid for insoluble Aβ (B). Samples were analyzed by ELISA, and Aβ values were normalized for total protein loaded and to vehicle-treated animals. To quantify plaque load by immunohistochemistry, 9 hemicoronal sections/animal were immunostained for 6E10, and plaque area analysis was performed as described under “Experimental Procedures.” TG, transgenic; Veh, vehicle. C, representative images from the cortex. The Aβ plaque area was quantitated in the cortex (D) and hippocampus (E) (n = 7–11 animals/group). *, p < 0.05; **, p < 0.01 compared with vehicle-treated APP/PS1 mice; Student's t test.

FIGURE 5.

Nuclear receptor agonists promote microglial colocalization with plaques. A, hemicoronal sections from each treatment group were coimmunostained with Iba1 (green) for microglia and 6E10 (red) for Aβ plaques. Representative images from the cortex are shown. TG, transgenic; Veh, vehicle. B and C, colocalization between 6E10 and Iba1 was analyzed as described under “Experimental Procedures” in the cortex (B) and hippocampus (C). n = 7–11 animals/group; *, p < 0.05; **, p < 0.01 compared with vehicle-treated APP/PS1 mice; Student's t test.

FIGURE 6.

Nuclear receptor agonists ameliorate cognitive deficits in AD mice. Training for the fear conditioning assay was performed on day 8 of drug treatment, and mice were tested on day 9. A, the number of freezes by each treatment group during training is shown as a function of periods. #, p < 0.05; ###, p < 0.001 between non-transgenic (TG) control mice and transgenic vehicle (Veh) mice by two-way ANOVA. B, the percentage of time spent freezing by each treatment group during the contextual fear conditioning test period. *, p < 0.05 compared with vehicle-treated APP/PS1 mice; #, p < 0.05 compared with non-transgenic littermate control mice by one-way ANOVA with Tukey post-test; n = 7–11 animals/group.