Fig 1.
Simplified representation of the pathways of brain cholesterol input, output, and storage along with potential mechanisms regulating these pathways. The pathways of major and minor quantitative significance are in black bold and gray bold, respectively. Not all the proteins in these pathways and regulatory mechanisms are indicated, mostly those that were studied or discussed in the present work. Different regulatory mechanisms are indicated in different colors. Arrows indicate activation, blunt-ends denote repression or inhibition. Protein symbols are deciphered in S1 Text. 24OH, 24-hydroxycholesterol, the product of CYP46A1; 27OH, 27-hydroxycholesterol, the product of CYP27A1.
Table 1.
Peptides and transitions for quantification of ABCA1 and HDACs by mass spectrometry.
Fig 2.
Sterol quantifications in the brain of Cyp46a1-/- mice and wild type controls.
Data for cholesterol, lathosterol, desmosterol, 24-hydroxycholesterol, and 27-hydroxycholesterol were taken from [49]. The data represent the mean ± SD of three measurements in individual (n = 3) 3–5 month old mice. Statistical significance was assessed two-way ANOVA followed by pairwise comparisons made using the Bonferroni correction. Significant comparisons of free sterols are denoted by the following color code: pink asterisks, significant changes between Cyp46a1-/- females versus wild type females; blue asterisks, significant changes between Cyp46a1-/- males versus wild type males; gray asterisks, significant changes between female and male mice of the same strain; and black asterisks, significant changes between the genotypes when data were collapsed across genders. *, P≤0.05; **, P≤0.01; ***, P≤0.001; and ND, not detectable (the limit of detection was 1 pmol/mg protein).
Fig 3.
Profiling of cholesterol-related genes in the Cyp46a1-/- brain by PCR array.
A) Female 3–4 month old mice. B) Male 3–4 month old mice. For both female and male mice, a pooled sample of 3 brain hemispheres from different mice was used. Each circle indicates an individual gene in the PCR array. The up-regulated and down-regulated genes are in green and red, respectively (cutoff, 2.0, dashed line), and those with Ct numbers ≤ 30 are in bold. Genes in blue are those which were downregulated in both genders. The number in parenthesis indicates a fold change as compared to the wild type brain. Gene abbreviations are deciphered in S1 Text.
Fig 4.
Evaluation of gene expression in the Cyp46a1-/- brain by qRT-PCR.
Genes are grouped based on function of the encoded proteins in cholesterol homeostasis. Each bar represents the mean ± SD of the PCR reactions performed individually on 3 to 6 animals in duplicate. The color code is the same as in Fig 2. Black asterisks indicate statistical significance between Cyp46a1-/- and wild type mice of the same gender as assessed by a two-tailed, unpaired Student’s t-test. *, P≤0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001; and *****, P≤0.00001.
Fig 5.
Quantification of proteins in the Cyp46a1-/- brain.
A) and B) Representative Western blots for SREBF2 (n = 3 female mice 3–4 months of age) and HMGCR (n = 3 male mice 6 months of age). α-Tubulin and β-actin were used for normalizations of SREBF2 and HMGCR, respectively. WT, wild type mice, KO, Cyp46a1-/- mice, MW, molecular weight standards. C) Mass spectrometry quantifications of ABCA1 and HDACs. Each bar represents the mean ± SD of individual measurements in 3 female mice of 3 months of age; the color code is the same as in Fig 2. Three to four transitions per peptide (two peptides for HDAC4 and one peptide for all other isoforms) were used. Black asterisks indicate statistical significance between Cyp46a1-/- and wild type mice as assessed by a two-tailed, unpaired Student’s t-test. *, P≤0.05.
Table 2.
Ubiquinated peptides with differential abundance in the Cyp46a1-/- brain as compared to the wild type brain.
Table 3.
Phosphoproteins with different phosphopeptide abundance in the Cyp46a1-/- brain as compared to the wild type brain.