Abstract
Cholesterol and 24-hydroxycholesterol are the most abundant brain sterols and represent the substrate and product, respectively, of cytochrome P450 46A1 (CYP46A1), a CNS-specific enzyme. CYP46A1 controls cholesterol elimination and turnover in the brain, the two processes that determine the rate of brain sterol flux through the plasma membranes and thereby the properties of these membranes. Brain sterol flux is decreased in Cyp46a1−/− mice compared to wild-type mice and increased in 5XFAD mice (a model of Alzheimer’s disease) when they are treated with a small dose of efavirenz, a CYP46A1 activator. Herein, we first assessed the brain proteome (synaptosomal fractions) and phospho-proteome (synaptosomal fractions and brain homogenates) of efavirenz-treated and control 5XFAD mice. Then, based on the pattern of protein abundance change, we conducted acetyl-CoA measurements (brain homogenates and mitochondria) and metabolic profiling (brain homogenates). The phospho-proteomics datasets were used for comparative analyses with the datasets obtained by us previously on mice with the same changes (efavirenz-treated and control 5XFAD mice from a different treatment paradigm) or with changes in the opposite direction (Cyp46a1−/− vs wild-type mice) in brain sterol flux. We found that CYP46A1 activity or the rate of brain sterol flux affects acetyl-CoA-related metabolic pathways as well as phosphorylation of cytoskeletal and other proteins. Knowledge of the key roles of acetyl-CoA and cytoskeletal phosphorylation in cell biology expands our understanding of the significance of CYP46A1-mediated cholesterol 24-hydroxylation in the brain and provides an additional explanation for why CYP46A1 activity modulations are beneficial in mouse models of different brain diseases.
Highlights
GC Gas chromatography LC Liquid chromatography MS Mass spectrometry tricarboxylic acid cycle (TCA) Tricarboxylic acid cycle UHPLC-MS/MS Ultra-high-performance liquid chromatography tandem mass spectrometry
Synaptosomal fractions rather than brain homogenates were used because our previous studies of brain homogenates by the label-free approach were not successful, possibly because the brain is very rich in lipids, which interfere with protein extraction and subsequent processing
EFV-treated 5XFAD mice, whose brain sterol flux was increased [13], had an ~ threefold increase in the abundance of ACADM, ACSS1 and MCCC1, the enzymes involved in acetyl-CoA biosynthesis, as well as increased expression of GYKL1, PGM2, PGM2L1, HK1, HK2 and PFKP, the enzymes that provide substrates for the synthesis of acetyl-CoA (Fig. 4)
Summary
GC Gas chromatography LC Liquid chromatography MS Mass spectrometry TCA Tricarboxylic acid cycle UHPLC-MS/MS Ultra-high-performance liquid chromatography tandem mass spectrometry. No results have been published yet, but there is a press release (https://ovidrx.com/science/clinical-studies/) stating that the study achieved its primary endpoint—a reduction in seizure frequency from the baseline This laboratory discovered CYP46A1 activation by pharmacologic means, namely, by EFV, and tested two paradigms of EFV treatment in 5XFAD mice, a model of Alzheimer’s disease [7, 13, 16, 17]. In the 1TP, EFV administration began when animals were still young and had not yet developed amyloid plaques in the brain Both treatments activated CYP46A1, enhanced brain cholesterol turnover, improved mouse performance in behavioural tests and had differential effects on the amyloid β load and expression of synaptic proteins as well as markers of inflammation [7, 13]. Altered sterol flux can change the activity and targeting of membrane-associated protein kinases or protein phosphatases to their protein substrates and vice versa and thereby modulate the extent of protein phosphorylation [18]
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