Cholesterol-metabolizing enzyme cytochrome P450 46A1 as a pharmacologic target for Alzheimer's disease

Abstract

Cytochrome P450 46A1 (CYP46A1 or cholesterol 24-hydroxylase) controls cholesterol elimination from the brain and plays a role in higher order brain functions. Genetically enhanced CYP46A1 expression in mouse models of Alzheimer's disease mitigates the manifestations of this disease. We enhanced CYP46A1 activity pharmacologically by treating 5XFAD mice, a model of rapid amyloidogenesis, with a low dose of the anti-HIV medication efavirenz. Efavirenz was administered from 1 to 9 months of age, and mice were evaluated at specific time points. At one month of age, cholesterol homeostasis was already disturbed in the brain of 5XFAD mice. Nevertheless, efavirenz activated CYP46A1 and mouse cerebral cholesterol turnover during the first four months of administration. This treatment time also reduced amyloid burden and microglia activation in the cortex and subiculum of 5XFAD mice as well as protein levels of amyloid precursor protein and the expression of several genes involved in inflammatory response. However, mouse short-term memory and long-term spatial memory were impaired, whereas learning in the context-dependent fear test was improved. Additional four months of drug administration (a total of eight months of treatment) improved long-term spatial memory in the treated as compared to the untreated mice, further decreased amyloid-β content in 5XFAD brain, and also decreased the mortality rate among male mice. We propose a mechanistic model unifying the observed efavirenz effects. We suggest that CYP46A1 activation by efavirenz could be a new anti-Alzheimer's disease treatment and a tool to study and identify normal and pathological brain processes affected by cholesterol maintenance.

Keywords: 24-Hydroxycholesterol; Alzheimer's disease; Amyloid; CYP46A1; Cholesterol; Efavirenz.

Conflict of interest statement

Conflict of interests

The authors have declared that no conflict of interest exists.

Copyright © 2017 Elsevier Ltd. All rights reserved.

Figures

Fig. 1. Sterol profiles in the brain of B6SJL and 5XFAD mice, EFV untreated (Utx) or treated (Tx)

Age-dependent changes were monitored in 1- to 9-month old animals. Gray asterisks are significant changes in Utx 5XFAD mice vs the background B6SJL strain; black asterisks are significant changes in Tx 5XFAD mice vs Utx 5XFAD mice. The results are mean ± SD of the measurements in individual animals (n=3–7 male mice per group and time point). *, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001 by repeated measures two-way ANOVA followed by a post hoc Bonferroni multiple comparison test. (M-P) To clearly see the overlaid sterol profiles, the statistical significance data (asterisks) are not indicated in these panels.

Fig. 2. CYP46A1 activation reduces dense-core amyloid plaques in 5-month old EFV-treated mice

A) Representative images of the brain sections (7–10 male mice per group) stained with ThioS (green). Nuclei were detected by propidium iodide and false colored in blue. B6SJL mice served as a negative control. Scale bars are 1 mm. B) Quantification of the Thioflavin S-positive plaques. Error bars indicate SD. *P ≤ 0.05 by a two-tailed, unpaired Student’s t-test.

Fig. 3. CYP46A1 activation reduces diffuse-core amyloid plaques and microglia activation in 5- and 9-month old EFV treated mice

A) Representative images of the brain sections (3–4 male mice per group) showing the anti-6E10 immunoreactivity (red) for diffuse-core amyloid plaques and anti-Iba1 immunoreactivity (green) for activated microglia. Nuclei were stained with DAPI. B6SJL mice served as a negative control. Scale bars are 1 mm. B) Quantification of the 6E10-positive amyloid plaques and Iba1-positive cells. Error bars indicate SD. C and D) Quantification of soluble and insoluble amyloid-β peptides by ELISA. Error bars indicate SD. Only males (n=12–13, colored in blue) were used for the measurements in 5 month-old animals; both males (n=4–5) and females (n=4, colored in pink) were used for the measurements in 9 month-old animals. Rectangles and spheres denote amyloid-β1–42 and amyloid-β1–40 peptides, respectively. Black asterisks are significant changes between the groups comprised of both males and females; blue asterisks are significant changes between the groups comprised of males only; pink asterisks are significant changes between the groups comprised of females only. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001 by a two-tailed, unpaired Student’s t-test.

Fig. 4. EFV effects on behavior in 5- and 9-month old 5XFAD mice

Utx, untreated animals; Tx, treated animals; US, unconditioned stimulus; and CS, conditioned stimulus. Bars represent mean ± SEM. A and D) *, P ≤ 0.05 by a two-tailed, unpaired Student’s t-test. B and C) *, P ≤ 0.05; and ***, P ≤ 0.001 by a two-way ANOVA with treatment group and trial factors, followed by a Bonferroni corrections as a post hoc comparison if needed.

Fig. 5. Effects of CYP46A1 activation on APP and gene expression

A and D) Representative Western blots (n=6–8 mice per group of 5- and 9-month old males) showing the reduction in the brain levels of APP. The relative APP expression is presented below the Western blot and is normalized per β-actin. At 5 months of age, the APP reduction was statistically significant, P = 0.03 by a two-tailed, unpaired Student’s t-test. At 9 months of age, the reduction was not significant, P = 0.15, and only represents a trend. B) The Volcano plot showing the reductions in the gene expression of EFV-treated vs untreated mice as indicated by mRNA sequencing of the whole brain transcriptome. The vertical dashed lines are the boundaries of a 2.5-fold change in gene expression (an arbitrary cut off); the horizontal dashed line is the boundary of the P value of 0.05. Genes with ≥ 2.5-fold change and q ≤ 0.05 are indicated, and those of pertinence to AD are also colored in orange. Three mice per group were used as biological replicates; all 6 were littermates. C) Quantifications by qPCR confirming a reduction in gene expression in EFV-treated vs untreated 5XFAD mice indicated by the transcriptome sequencing. The results are mean ± SD of the measurements in individual mice. E) Representative Western blot (n=7 mice per group) showing the expression of the APP cleavage fragments C99 and C83. The relative fragment expression is normalized per GAPDH. (F) Quantifications of the C99 and C83 fragments: individual, as a sum (C83 + C99), and as the ratio (C83/C99). Error bars indicate SD. *, P ≤ 0.05 by a two-tailed, unpaired Student’s t-test.

Fig. 6. Proposed model unifying EFV effects

The sequence of events is numbered chronologically with primes (′, ″, ‴) indicating events that may occur simultaneously. Events in black above black arrows are those supported experimentally by the present work; events in gray above gray arrows are putative and suggested, in most cases, on the basis of literature data. Thin and thick black lines underline events after 4- and 8-months of EFV treatment, respectively. Events 1 and 2: CYP46A1 is the only enzyme in mice that can produce 24HC in the brain (Lund et al., 1999), hence an increase in the brain 24HC levels measured in Fig. 1D–P provides evidence for enzyme activation by EFV. Event 3 is supported by an increase in the brain lathosterol levels triggered by an increase in the 24HC levels (Fig. 1B–N). Events 3′ and 4 are supported by a decrease in the APP levels in EFV-treated mice (Fig. 5A,B) as well as the literature data summarized in Discussion ((Brown et al., 2004; Chia and Gleeson, 2011; Simons et al., 1998). Events 3″ and 4′ are supported by improved performance of EFV-treated mice after 8 months of drug treatment in Morris water maze tasks (Fig. 4B) and also literature data presented in Discussion (Kotti et al., 2008; Kotti et al., 2006; Paul et al., 2013; Sun et al., 2016a). Event 3′″ cannot be measured in vivo directly, only indirectly by measuring the expression of the target genes in cells of interest. Events 5, 5′, and 5″: neuroinflammation, a hallmark of Alzheimer’s disease brain, is characterized by the presence of activated astrocytes and microglia around amyloid plaques (Akiyama et al., 2000; Cameron and Landreth, 2010; Schwab and McGeer, 2008). LXRs are suggested to exert anti-inflammatory effects in multiple cell type and contexts by inhibiting the transcription of the NF-κB-regulated proinflammatory genes such as IL-1β and TNFα (Ghisletti et al., 2007). Event 5″ is supported by a reduction in brain microglia activation as a result of EFV treatment (Fig. 3A). Event 5 is supported by a lowered Serpina3k expression in the whole brain transcriptome of EFV-treated mice (Fig. 5B) and data that in Alzheimer’s disease brain and mouse models of this disease, plaque-surrounding activated astrocytes have elevated levels of α-1-antichymotrypsin encoded by SERPINA3 (Abraham et al., 1988; Licastro et al., 1998; Nilsson et al., 2001), a human ortholog of Serpina3k. Events 5′ and 6: Serpina1e and Serpina3k, the two acute phase genes, are downregulated in EFV-treated mice (Fig. 5B,C) indicating a decrease in inflammatory response in infiltrating brain macrophages and brain astrocytes, respectively (Schmechel and Edwards, 2012; Styren et al., 1998). This downregulation is also consistent with a reduction in brain microglia activation as a result of EFV treatment (Fig. 3A). Furthermore, Events 5′ and 6 are supported by a lowered Serpina3n expression in APP transgenic mice treated with the synthetic LXR agonist T0901317; these mice have a decrease in insoluble amyloid-β peptide along with a suppression of inflammatory response (Lefterov et al., 2007). The beneficial role of activated LXRs in amyloid pathology is also supported by an increase in amyloid plaque load in APP/presenillin transgenic mice lacking LXRs and showing no inhibition of the inflammatory response of glial cells to amyloid-β fibrils (Zelcer et al., 2007). Finally, Event 7 is supported by the literature data (Abraham and Potter, 1989; Eriksson et al., 1995; Ma et al., 1996; Ma et al., 1994), and Event 8 by amyloid burden reduction in EFV-treated mice (Figs. 3C,D).