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.

CYP46A1 activation by low-dose efavirenz enhances brain cholesterol metabolism in subjects with early Alzheimer’s disease

“…Mechanistically, CYP46A1 activation by EFV increased the rates of the mevalonate pathway as well as sterol flux through the plasma membranes and thereby altered physico-chemical properties of plasma membranes and membrane-dependent events, such as synaptic transmission and phosphorylation of cytoskeletal and other proteins [ 26 , 27 ]. In addition, increased sterol flux was shown to increase in 5XFAD mice total brain acetyl-CoA content, energetic state of brain mitochondria, and brain acetylcholine levels [ 27 , 28 ]. A model of how one enzyme can control multiple and apparently unrelated processes in the brain was proposed [ 29 , 30 ].…”

7,8-Dihydroxy Efavirenz Is Not as Effective in CYP46A1 Activation In Vivo as Efavirenz or Its 8,14-Dihydroxy Metabolite

“…We called these two sites EFV-and L-Glu-binding, respectively, and showed that when activators bind to each site simultaneously, their effect on CYP46A1 activity is synergistic [33,35]. We established that EFV monohydroxy metabolites can bind to both CYP46A1 allosteric sites, whereas 7,8-dihydroxy EFV [7,8diOH EFV, either (S) or (rac)] only interacts with EFV-binding sites, and 8,14-dihydroxy EFV [8,14diOH EFV, either (S) or (rac)] binds only to the L-Glu-binding site [31].…”

“…Herein, (rac)-7,8diOH EFV (called 7,8diOH EFV hereafter) was investigated to compare its brain effects with those of 8,14diOH EFV. We established that at a dose (0.1 mg/kg of body weight per day), delivery route (in drinking water), and treatment duration (6 months, from 3 to 9 months of age) which were similar to treatments with 8,14diOH EFV and (S)-EFV [16,31], 7,8diOH EFV also activated CYP46A1 in the brain of 5XFAD mice. However, the extent of the enzyme activation and other therapeutically relevant effects were not as pronounced as with (S)-EFV and 8,14diOH EFV.…”

“…Also, there were increased levels of brain acetyl-CoA and acetylcholine (Ach), altered gene expression, synaptic L-Glu release, abundance of synaptic proteins, and protein phosphorylation as well as decreased amyloid β (Aβ) load, either in the whole brain or specific brain regions. Accordingly, either individually or collectively, these treatment outcomes likely contribute, at least in part, to behavioral improvements in EFV-treated animals [11,16,30,31]. However, at higher doses (>0.22 mg/kg of body weight per day in mice) EFV inhibits CYP46A1 by binding to the enzyme active site, i.e., prevents cholesterol from entering the site of catalysis [27].…”

“…The in vitro activation and binding data raised a question regarding whether EFV hydroxymetabolites can activate CYP46A1 in vivo and lead to brain effects that are similar or even better than those of (S)-EFV. We already evaluated (rac)-8,14diOH EFV (called 8,14diOH EFV hereafter) on 5XFAD mice and obtained results suggesting that 8,14diOH EFV and (S)-EFV should be given to 5XFAD mice simultaneously to test whether such a co-administration will enhance the therapeutic effects of (S)-EFV [31]. Herein, (rac)-7,8diOH EFV (called 7,8diOH EFV hereafter) was investigated to compare its brain effects with those of 8,14diOH EFV.…”