Increased Acetylcholine Levels and Other Brain Effects in 5XFAD Mice after Treatment with 8,14-Dihydroxy Metabolite of Efavirenz

Abstract

Efavirenz (EFV), an FDA-approved anti-HIV drug, has off-target binding to CYP46A1, the CNS enzyme which converts cholesterol to 24-hydroxycholesterol. At small doses, EFV allosterically activates CYP46A1 in mice and humans and mitigates some of the Alzheimer's disease manifestations in 5XFAD mice, an animal model. Notably, in vitro, all phase 1 EFV hydroxymetabolites activate CYP46A1 as well and bind either to the allosteric site for EFV, neurotransmitters or both. Herein, we treated 5XFAD mice with 8,14-dihydroxyEFV, the binder to the neurotransmitter allosteric site, which elicits the highest CYP46A1 activation in vitro. We found that treated animals of both sexes had activation of CYP46A1 and cholesterol turnover in the brain, decreased content of the amyloid beta 42 peptide, increased levels of acetyl-CoA and acetylcholine, and altered expression of the brain marker proteins. In addition, male mice had improved performance in the Barnes Maze test and increased expression of the acetylcholine-related genes. This work expands our knowledge of the beneficial CYP46A1 activation effects and demonstrates that 8,14-dihydroxyEFV crosses the blood-brain barrier and has therapeutic potential as a CYP46A1 activator.

Keywords: 8,14-dihydroxyefavirenz; Alzheimer’s disease; CYP46A1; acetyl-CoA; acetylcholine; cholesterol metabolism; efavirenz.

Figure 1

8,14-DihydroxyEFV effect on sterol content in the brain. Data represent the mean ± SD of the measurements in individual 5XFAD mice (8 female and 8 male). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 as assessed by two-way ANOVA with Tukey’s multiple comparison test. The asterisk color indicates significance between female mice (pink), male mice (blue), or male-female animals (black). Cntr, control mice; Tx, treated mice.

Figure 2

8,14-DihydroxyEFV effect on the brain Aβ peptides. Data represent the mean ± SD of the measurements in individual 5XFAD mice (12 female and 12 male). *** p ≤ 0.001 as assessed by two-way ANOVA with Tukey’s multiple comparison test. The asterisk color indicates significance between female mice (pink) and male mice (blue). Cntr, control mice; Tx, treated mice.

Figure 3

8,14-DihydroxyEFV effect on performance of 5XFAD mice in behavioral tasks: (A), Barnes Maze test; (B), Y-maze test; and (C), fear conditioning tests. Data represent the mean ± SEM of the measurements in individual mice (17 control female mice, 15 treated female mice, 16 control male mice, and 17 treated male mice). Statistical significance was assessed by two-way repeated measures ANOVA with Bonferroni correction (the Barnes Maze test) and a two-tailed unpaired Student’s t-test (the Y-maze test and fear conditioning tests). * p = 0.03 was only detected between the session number and treatment factor in the Barnes maze test for male mice. Cntr, control mice; Tx, treated mice.

Figure 4

8,14-DihydroxyEFV effect on the brain acetyl-CoA levels. Data represent the mean ± SD of the measurements in individual mice (8 female mice and 8 male mice). *** p ≤ 0.001 as assessed by two-way ANOVA with Tukey’s multiple comparison test. The asterisk color indicates significance between female mice (pink), male mice (blue) or male-female animals (black). Cntr, control mice; Tx, treated mice.

Figure 5

8,14-DihydroxyEFV and EFV effects on the brain Ach levels. Data represent the mean ± SD of the measurements in individual mice (5–12 female mice and 5–12 male mice). *, p ≤ 0.05; ***, p ≤ 0.001 as assessed by two-way ANOVA with Tukey’s multiple comparison test. The asterisk color indicates significance between female mice (pink), male mice (blue) or male-female animals (black). Cntr, control mice; Tx, treated mice.

Figure 6

8,14-DihydroxyEFV effect on brain expression of Ach-related genes. Data represent the mean ± SD of the measurements in five individual male mice. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 as assessed by a two-tailed unpaired Student’s t-test. Cntr, control mice; Tx, treated mice.

Figure 7

CYP46A1 activation in vitro in the presence of different compounds. CYP46A1 activity is presented as nanomoles of 24-hydoxycholsterol (24HC) formed per nmole of CYP46A1 per min. The results are the mean ± SD of the measurements from the three independent experiments. *** p ≤ 0.001 as assessed by a two-tailed unpaired Student’s t-test. 8,14-diOH EFV, 8,14-dihydroxyEFV.

Figure 8

8,14-DihydroxyEFV (8,14-diOH EFV) effect on brain expression of various marker proteins. (A), Representative Western blots of brain homogenates. Each lane, except those with molecular weight markers, represents a sample from an individual animal. Of the four samples in each group, two left lanes are always samples from male mice and two right lanes are always samples from female mice. The Western blot for CYP46A1 represents expression in male mice only as a similar gel for female mice is not shown, although quantified in the panel below. Also not shown is a Western blot for CYP46A1 when two female and two male mice for each group were used and the data suggested lack of sex-based differences and the treatment effect. (B), Quantification of the relative protein expression in (A). Protein expression in each sample within a group was first normalized to the β-actin expression followed by the calculation of the mean value of the protein expression within a group. This mean value was then normalized to the mean value of the protein expression in control 5XFAD mice, which was taken as one. The results represent the mean ± SD of the measurements in individual mice. * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001 as assessed a two-tailed, unpaired Student’s test. Data for protein expression of EFV-treated mice are shown for comparison and are taken from [7].