ApoE4 disrupts sterol and sphingolipid metabolism in Alzheimer's but not normal brain

Abstract

The epsilon 4 allele of ApoE is associated with an earlier onset and faster progression of Alzheimer's disease in patients with the familial form of this neurodegenerative condition. Although ApoE4 has been repeatedly associated with altered sphingomyelin and cholesterol levels in tissue culture and rodent models, there has not been a direct quantification of sphingomyelin or sterol levels in the brains of patients with different forms of ApoE. We measured the sphingolipid and sterol content of human brain tissues and found no evidence of perturbed sterol or sphingolipid biochemistry in the brains of individuals expressing ApoE4 who did not have a preexisting neurodegenerative condition. Nevertheless, ApoE4 was associated with gross abnormalities in the sterol and sphingolipid content of numerous brain regions in patients with Alzheimer's disease. The findings suggest that ApoE4 may not by itself alter sterol or sphingolipid metabolism in the brain under normal conditions, but that other neuropathologic changes of Alzheimer's are required to unmask the effect of ApoE4, and to perturb sterol and sphingolipid biochemistry.

Figure 1. Sphingomyelin, ceramide and sterol levels are increased in the grey matter of middle frontal gyrus in AD brain

Total lipids were extracted from the MFG grey matter and sphingomyelin, ceramide and sterols were identified and quantified by ESI/MS/MS. In each panel, representative spectra are presented on the left, and bar graphs comparing quantification of constituents from AD and normal brain are on the right. (A) Spectra from MFG grey matter of an AD patient, showing the identification and exact mass of sphingomyelin C16:0, C18:0, C20:0, C22:0 and C24:0. Bar graph compares quantification of sphingomyelin constituents from total ApoE3 and ApoE4 in AD and normal brains. Increases in AD brain are: sphingomyelin C16:0 (1.8-fold); C18:0 (4.9-fold); C22:0 (4.4-fold); C24:0 (2.4-fold). (B) Spectra of ceramides from the MFG grey matter of an AD patient showing the exact mass of C16:0, C18:0, C22:0, C24:1, C24:0, steraoyl and sulfatide. Bar graph compares quantification of ceramides in AD and normal brain. Decreases in ceramide are: 24:0 (3.6-fold) and steraoyl (2.7-fold). (C) Spectrum from the MFG grey matter showing the identification and exact mass of free cholesterols and the cholesterol esters C16:0 and C18:1. Bar figures compare the quantification of cholesterol and cholesterol esters in AD and normal brain. Data are mean and S.D. of counts per second (cps). n = 30 patient samples per group. ** = p < 0.01 and *** = p < 0.001. ANOVA with Tukey post hoc comparisons.

Figure 2. Ceramide levels are decreased in white matter of middle frontal gyrus in AD brain

White matter was dissected from the middle frontal gyrus (MFG) of AD and normal brains and sterol, sphingomyelin and ceramide levels were determined by ESI/MS/MS. (A) Levels of free cholesterol (monomer, dimmer and trimer combined), (B) the cholesterol esters C16:0 and C18:1 (mono-, di- and trimer combined) and (C) sphingomyelins: none were significantly different in AD vs. normal brain. (D) Ceramides were separated by carbon chain length. Whereas C18:0 and C24:0 were not significantly different, there were significant decreases in ceramide C16:0 (3.7-fold), C22:0 (4.5-fold), C24:1 (9.2-fold), steraoyl (8.4-fold) and sulfatide (4.4-fold) in AD compared with normal brains. Data are mean and S.D. of counts per second (cps). n = 30 patient samples per group. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001. Statistical tests include: Students T-test for cholesterol and cholesterol ester and ANOVA with Tukey post hoc comparisons for sphingomeylins and ceramides.

Figure 3. ApoE4 is associated with increased sterol levels in grey matter of AD but not normal subjects

White and grey matter were separated from the MFG and MTG before extracting total lipids for analysis by ESI/MS/MS. (A) In the MFG, there were significant increases of cholesterol (2.6-fold), (B) cholesterol esters C16:0 (3.4-fold), and C18:1 (3.3-fold), and (C) in the MTG, the cholesterol ester C18:1 (2.4-fold), in ApoE4 compared with ApoE3 AD brain. (D) In the MFG, cholesterol, (E) cholesterol esters, and (F) in the MTG cholesterol esters, were similar in ApoE4 compared with ApoE3 normal brain. Graphs are summary data of cholesterol (mono-, di- and trimer) and the indicated cholesterol esters (mono-, di- and trimer). Data are mean and S.D. of counts per second (cps). n = 15 patient samples per group. *** = p < 0.001. Students T-test.

Figure 4. ApoE4 is associated with depletion of sphingomyelins and accumulation of ceramides in AD but not in normal subjects

White and grey matter were dissected from the MFG of AD and normal brain. Total lipids were extracted for identification of sphingomyelins and ceramides by ESI/MS/MS. (A) In the MFG grey matter, there were significant decreases of sphingomyelin C22:0 (12.5-fold), and C24:0 (6.2-fold), and (B) significant increases of ceramide C18:0 (2.0-fold) and C24:1 (1.8-fold) in ApoE4 compared with ApoE3 AD brain. (E) In the MFG white matter, while there were no significant differences in sphingomeylin, there were significant differences in ceramide C22:0 (2.6-fold increase), C24:0 (1.2-fold decrease), and sulfatide (1.7-fold increase), in ApoE4 vs. ApoE3 brain. (E) In the MFG grey matter there were no significant differences in sphingomyelin or (F) ceramide, and (G) in the MFG white matter there were no differences in sphingomyelin or (H) ceramide of ApoE4 compared with ApoE4 normal brain. Data are mean and S.D. of counts per second (cps). n = 15 patient samples per group. ** = p < 0.01 and *** = p < 0.001. ANOVA with Tukey post hoc comparisons for sphingomyelins and ceramides.

Figure 5. 4-hydroxynonenal is increased in the frontal grey matter of AD patients expressing ApoE4, but not in normal subjects expressing one or more ApoE4 alleles

The MFG of AD and normal brain was dissected into white and grey matter and 4-HNE was measured by ESI/MS/MS. The lysine and histadine adducts of 4-HNE are shown. (A) In the MFG grey matter there was a significant increase in the lysine adduct of 4-HNE (4.9-fold) and, (B) no significant difference in MFG white matter content of ApoE4 compared with ApoE3 AD brain. (C) In the MFG grey, (D) and MFG white matter, there were no significant differences in 4-HNE of ApoE4 compared with ApoE3 normal brain. Data are mean and S.D. of counts per second (cps)n = 15 patient samples per group. *** = p< 0.001. Students T-test.