ACAT1 gene ablation increases 24(S)-hydroxycholesterol content in the brain and ameliorates amyloid pathology in mice with AD

Abstract

Cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative diseases, including the abnormal accumulation of amyloid-beta, one of the pathological hallmarks of Alzheimer disease (AD). Acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) are two enzymes that convert free cholesterol to cholesteryl esters. ACAT inhibitors have recently emerged as promising drug candidates for AD therapy. However, how ACAT inhibitors act in the brain has so far remained unclear. Here we show that ACAT1 is the major functional isoenzyme in the mouse brain. ACAT1 gene ablation (A1-) in triple transgenic (i.e., 3XTg-AD) mice leads to more than 60% reduction in full-length human APPswe as well as its proteolytic fragments, and ameliorates cognitive deficits. At 4 months of age, A1- causes a 32% content increase in 24-hydroxycholesterol (24SOH), the major oxysterol in the brain. It also causes a 65% protein content decrease in HMG-CoA reductase (HMGR) and a 28% decrease in sterol synthesis rate in AD mouse brains. In hippocampal neurons, A1- causes an increase in the 24SOH synthesis rate; treating hippocampal neuronal cells with 24SOH causes rapid declines in hAPP and in HMGR protein levels. A model is provided to explain our findings: in neurons, A1- causes increases in cholesterol and 24SOH contents in the endoplasmic reticulum, which cause reductions in hAPP and HMGR protein contents and lead to amelioration of amyloid pathology. Our study supports the potential of ACAT1 as a therapeutic target for treating certain forms of AD.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

ACAT protein, enzyme activity, and RNA expression in mouse brains. (A) ACAT activity in 53-d-old mouse brain homogenates and (B) ACAT activities in various regions. Cereb, cerebellum; BrStm, brainstem; Cx, cortex; Th, thalamus; Hp, hippocampus. (C) Immunodepletion of ACAT activity in the WT mouse brain homogenates. IP with nonspecific rabbit IgG or with ACAT1-specific (A1) IgG. The ACAT activities in the supernatants were measured. (D) Identification of A1 protein. After IP, pellets were resolved by SDS/PAGE; A1 protein (46 kDa) was detected with polyclonal A1 antibodies. Lysates from WT and A1− mouse adrenals were used as controls. (E) A1 mRNA distribution. Upper: Nissl staining from a 2-month-old WT mouse. Cx, cortex; Hp, hippocampus; Am, amygdale. (Middle and Bottom) In situ hybridizations using [32P]-ACAT1 antisense riboprobe or sense riboprobe (as negative control). For bottom panel, brain periphery was outlined artificially. (Scale bar: 250 mm.) (F) A1 mRNA levels in WT mouse hippocampus and cortex as measured by real-time PCR and normalized against neurofilament polypeptide chain (NF120) mRNA. Data in A–D and F represent mean ± SEM; n = 2.

Fig. 2.

Effect of A1− on Aβ pathology in AD mice. (A) Intraneuronal Aβ (using human Aβ-specific antibody 6E10) in the hippocampal CA1 region of male mice at 4 months (P = 0.0059; n = 4 or 5). (B) Aβ42 and Aβ40 levels analyzed by ELISA in the forebrains of mice at 17 months. For Aβ42, P = 0.035; for Aβ40, P = 0.084; n = 5. (C) Amyloid plaque load (using thioflavin S staining) in the hippocampus of 17-month-old mice; P = 0.031; n = 5. (Scale bars: 100 μm in A and C.) Data represent mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 3.

Effect of A1− on human and mouse APPs (mAPP) and their cleavage products in AD and NTG mouse brains. (A) Immunoblot analysis and (B–D) quantification. For hAPP, P = 0.042; for hCTFβ, P = 0.017; for hsAPPα, P = 0.004. Forebrains of 4-month-old mice were used; n = 7. (E) mRNA analysis of hAPP gene by real-time PCR; n = 6. (F) Immunoblot analysis and (G and H) quantification of mature and immature forms of hAPP from 25-d-old AD mouse forebrains; P = 0.019 (G) and P = 0.046 (H); n = 9. (I) For the ratio of mature to immature hAPP, P = 0.368; n = 9. (J) Immunoblot analysis and quantitation of mouse and human APP (m+hAPP) from 4-month-old NTG and A1/AD mouse forebrains. Full-length m+hAPP is detected by using antiserum 369, which recognizes both mouse and human APP (n = 7). (K) Immunoblot analysis and quantitation of mouse endogenous APP (mAPP) and its cleavage fragments from 2-month-old C57BL/6 mouse forebrains. Antiserum 369 was used to detect mAPP and the CTF fragments (mCTFβ and mCTFα). P = 0.168 for mAPP, P = 0.605 for mCTFβ, and P = 0.504 for mCTFα; n = 3. Data represent mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 4.

Sterol metabolism in A1+/AD and A1−/AD mouse forebrains; 4-month-old male mice were used. (A) GC-MS analysis of cholesterol (CHOL); P = 0.04; n = 5. (B) GC-MS analysis of lanosterol (LAN), desmosterol (DES), and 24SOH. For 24SOH, P = 0.007; n = 5. (C) Sterol synthesis in vivo (P = 0.04); n = 9. (D) Fatty acid synthesis in vivo; n = 9. (E) Esterification of [3H]cholesterol in mouse brains; P = 0.0009; n = 6. (F) Immunoblot analysis and (G) quantification of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR); P = 0.001; n = 6. (H) Relative expression of HMGR mRNA analyzed by real-time PCR; P = 0.71; n = 6. (I) Relative expressions of mRNAs of SRE response genes or LXR response genes analyzed by real-time PCR. HMGR, P = 0.71; 3-hydroxy-3-methylglutaryl CoA synthase (HMGS), P = 0.24; squalene synthase (SQS), P = 0.48; lipoprotein receptor-related protein-1 (LRP), P = 0.35; LDL receptor (LDLR), P = 0.91; sterol regulatory element–binding protein 2 (SREBP2), P = 0.35; sterol regulatory element–binding protein 1 (SREBP1), P = 0.54; apolipoprotein E (APOE), P = 0.07 (i.e., the difference approached but did not reach statistical significance); ATP-binding cassette transporter subfamily A member 1 (ABCA1), P = 0.27; ABCG1, P = 0.058; ABCG4, P = 0.63; cytochrome P450 46A1 (CYP46A1), P = 0.3; n = 6. Data represent mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001.

Fig. 5.

Biosynthesis and regulatory activities of 24SOH in primary hippocampal neurons. For A–E, hippocampal neurons from A1+/AD and A1−/AD mice were employed. (A) Cholesterol esterification in intact cells. Cells were cultured for 7 d; lipids in cells were extracted and analyzed by TLC; P = 0.037. (B and C) Biosynthesis of [3H]sterols from [3H]acetate. Cells were cultured for 14 d. The lipids were analyzed by TLC. (B) [3H]Cholesterol (CHOL), P = 0.4 for media and P = 0.05 for cells and media. (C) [3H]24(S)-hydroxycholesterol (24SOH), P = 0.04 for media and P = 0.01 for cells and media. (D) Secretion of newly synthesized CHOL (P = 0.38) and 24SOH (P = 0.19); n = 2. (E) Immunoblot analysis of CYP46A1. Cells were cultured for 20 d; n = 2. (F) Immunoblot analysis of hAPP and HMGR in A1+/AD hippocampal neurons incubated with 1 μM 24SOH [delivered in ethanol (EtOH) at 0.1%] for 0.5 to 3 h. Cells were cultured for 35 d; (G) Effects of treating A1+/AD hippocampal neurons with 1 μM or 5 μM of 24SOH for 3 h on hAPP and HMGR levels. For 5 μM 24SOH, P = 0.0003 for hAPP and P = 0.03 for HMGR. Cells were cultured for 4 or 8 weeks; n = 3. For E–G, values were normalized against the β-actin signal in each lane. (H) Relative expression of hAPP mRNA by real-time PCR; n = 3. Data represent mean ± SEM. *P < 0.05; **P < 0.01.