ATP-binding cassette transporter A1 mediates the beneficial effects of the liver X receptor agonist GW3965 on object recognition memory and amyloid burden in amyloid precursor protein/presenilin 1 mice

Abstract

The cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) moves lipids onto apolipoproteins including apolipoprotein E (apoE), which is the major cholesterol carrier in the brain and an established genetic risk factor for late-onset Alzheimer disease (AD). In amyloid mouse models of AD, ABCA1 deficiency exacerbates amyloidogenesis, whereas ABCA1 overexpression ameliorates amyloid load, suggesting a role for ABCA1 in Aβ metabolism. Agonists of liver X receptors (LXR), including GW3965, induce transcription of several genes including ABCA1 and apoE, and reduce Aβ levels and improve cognition in AD mice. However, the specific role of ABCA1 in mediating beneficial responses to LXR agonists in AD mice is unknown. We evaluated behavioral and neuropathogical outcomes in GW3965-treated female APP/PS1 mice with and without ABCA1. Treatment of APP/PS1 mice with GW3965 increased ABCA1 and apoE protein levels. ABCA1 was required to observe significantly elevated apoE levels in brain tissue and cerebrospinal fluid upon therapeutic (33 mg/kg/day) GW3965 treatment. At 33 mg/kg/day, GW3965 was also associated with a trend toward redistribution of Aβ to the carbonate-soluble pool independent of ABCA1. APP/PS1 mice treated with either 2.5 or 33 mg/kg/day of GW3965 showed a clear trend toward reduced amyloid burden in hippocampus and whole brain, whereas APP/PS1-treated mice lacking ABCA1 failed to display reduced amyloid load in the whole brain and showed trends toward increased hippocampal amyloid. Treatment of APP/PS1 mice with either dose of GW3965 completely restored novel object recognition memory to wild-type levels, which required ABCA1. These results suggest that ABCA1 contributes to several beneficial effects of the LXR agonist GW3965 in APP/PS1 mice.

FIGURE 1.

ABCA1 protein levels reflect GW3965 dose. Cortical and hippocampal extracts from untreated (−: black) and treated (+: white) mice were immunoblotted for ABCA1. Left panels are representative blots using undiluted samples from individual mice from the following cohorts: APP/PS1 untreated control: n = 7 (cortex), n = 7 (hippocampus); APP/PS1 prophylactic: n = 8 (cortex) n = 5 (hippocampus); APP/PS1 low dose therapeutic: n = 8 (cortex), n = 6 (hippocampus); APP/PS1 high dose therapeutic: n = 10 (cortex), n = 9 (hippocampus); ABCA1^−/− APP/PS1 untreated control: n = 4 (cortex), n = 4 (hippocampus); ABCA1^−/− APP/PS1 prophylactic: n = 8 (cortex) n = 8 (hippocampus): ABCA1^−/− APP/PS1 low dose therapeutic: n = 8 (cortex), n = 8 (hippocampus); ABCA1^−/− APP/PS1 high dose therapeutic: n = 4 (cortex), n = 4 (hippocampus). Samples from each mouse were normalized to actin and expressed as fold-difference compared with ABCA1 levels in untreated APP/PS1 controls. Data represent mean ± S.E., where * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001 by Students t test.

FIGURE 2.

High dose GW3965 is required for elevated apoE levels in tissue. Cortical and hippocampal extracts from APP/PS1 untreated (−: white), APP/PS1 treated (+: dark gray), APP/PS1 ABCA1^−/− untreated (−: light gray), and APP/PS1 ABCA1^−/− treated (+: black) mice were immunoblotted for apoE. Left panels are representative blots using undiluted samples from individual mice from the following cohorts: APP/PS1 untreated control: n = 7 (cortex), n = 8 (hippocampus); APP/PS1 prophylactic: n = 8 (cortex) n = 8 (hippocampus); APP/PS1 low dose therapeutic: n = 8 (cortex), n = 8 (hippocampus); APP/PS1 high dose therapeutic: n = 10 (cortex), n = 9 (hippocampus); ABCA1^−/− APP/PS1 untreated control: n = 4 (cortex), n = 4 (hippocampus); ABCA1^−/− APP/PS1 prophylactic: n = 8 (cortex) n = 8 (hippocampus): ABCA1^−/− APP/PS1 low dose therapeutic: n = 7 (cortex), n = 6 (hippocampus); ABCA1^−/− APP/PS1 high dose therapeutic: n = 4 (cortex), n = 4 (hippocampus). Samples from each mouse were blotted at least twice, normalized to actin, and expressed as fold-difference compared with apoE levels in untreated APP/PS1 controls. Data represent mean ± S.E., where * represents p < 0.05 and ** represents p < 0.01 by one-way analysis of variance followed by Tukey post test.

FIGURE 3.

ABCA1 is required for increased CSF apoE levels in response to GW3965. Equal volumes of CSF from individual mice from untreated control (C), prophylactic (P), low dose therapeutic (T), and high dose therapeutic (TH) groups of APP/PS1 and APP/PS1 ABCA1^−/− mice were pooled, separated by 6% native PAGE, and immunoblotted for apoE and albumin. Stokes diameter markers are shown on the right.

FIGURE 4.

Effect of GW3965 and ABCA1 on cortical and hippocampal Aβ levels. Tissues were serially extracted with carbonate followed by guanidine hydrochloride and Aβ40 and Aβ42 levels in each fraction were measured by ELISA. Data represent mean ± S.E. of 1–4 measurements from each mouse. Cohorts consisted of the following Ns: APP/PS1 untreated control: n = 7 (cortex), n = 8 (hippocampus); APP/PS1 prophylactic: n = 8 (cortex) n = 8 (hippocampus); APP/PS1 low dose therapeutic: n = 8 (cortex), n = 8 (hippocampus); APP/PS1 high dose therapeutic: n = 10 (cortex), n = 10 (hippocampus); ABCA1^−/− APP/PS1 untreated control: n = 4 (cortex), n = 4 (hippocampus); ABCA1^−/− APP/PS1 prophylactic: n = 8 (cortex) n = 8 (hippocampus): ABCA1^−/− APP/PS1 low dose therapeutic: n = 8 (cortex), n = 8 (hippocampus); ABCA1^−/− APP/PS1 high dose therapeutic: n = 4 (cortex), n = 4 (hippocampus). Data represent mean ± S.E., where * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001 by one-way analysis of variance followed by Tukey post test. C, control; P, prophylactic; T, low dose therapeutic; TH, high dose therapeutic.

FIGURE 5.

High dose GW3965 solubilizes amyloid in APP/PS1 mice with functional ABCA1. top, thioflavin-S staining of hemicoronal sections from APP/PS1 mice in the presence or absence of ABCA1, with or without GW3965 treatment. Scale bar represents 1000 μm. Amyloid load was quantitated in the hippocampus (bottom left) and whole hemi-brain (bottom right) as described under “Experimental Procedures.” Values represent mean ± S.E. of total amyloid load from 6 hemi-coronal brain sections per mouse, n = 2–6 mice per group. C, control; P, prophylactic; T, low dose therapeutic; TH, high dose therapeutic.

FIGURE 6.

ABCA1 is required for LXR-mediated improvement in object but not spatial memory. A–C, percentage of NOR in APP/PS1 mice with and without ABCA1 treated prophylactically with GW3965 for 24 weeks (A) or treated therapeutically at a low dose (B) or therapeutically at a high dose (C) for 8 weeks. Littermate controls include wild-type and untreated APP/PS1 animals. N represents the novel object, and cohort size is listed below the column. D–F, latency to locate a hidden platform in the MWM task in the same animals treated prophylactically (D) or therapeutically at a low dose (E) or therapeutically at a high dose (F) with GW3965. Columns represent the mean ± S.D. and, for the MWM task, are grouped into 4-day trial blocks. Cohort size for the MWM task are: wild-type, n = 8; untreated APP/PS1, n = 19; 24 weeks (prophylactic) APP/PS1 treated, n = 11; 24 weeks (prophylactic) APP/PS1/ABCA1^−/− treated, n = 8; 8 weeks (therapeutic) APP/PS1 treated, n = 11; 8 weeks (therapeutic) APP/PS1/ABCA1^−/− treated, n = 11. *** represents p < 0.0001; ** represents p < 0.01; * represents p < 0.05 comparing APP/PS1 treated to wild-type littermate controls. +++ represents p < 0.001; ++ represents p < 0.01; + represents p < 0.05 comparing APP/PS1/ABCA1^−/− to wild-type littermate controls. C, control; T, low dose therapeutic.