Differential effects of 24-hydroxycholesterol and 27-hydroxycholesterol on beta-amyloid precursor protein levels and processing in human neuroblastoma SH-SY5Y cells

Abstract

Background: Activation of the liver x receptors (LXRs) by exogenous ligands stimulates the degradation of beta-amyloid 1-42 (Abeta42), a peptide that plays a central role in the pathogenesis of Alzheimer's disease (AD). The oxidized cholesterol products (oxysterols), 24-hydroxycholesterol (24-OHC) and 27-hydroxycholesterol (27-OHC), are endogenous activators of LXRs. However, the mechanisms by which these oxysterols may modulate Abeta42 levels are not well known.

Results: We determined the effect of 24-OHC and/or 27-OHC on Abeta generation in SH-SY5Y cells. We found that while 27-OHC increases levels of Abeta42, 24-OHC did not affect levels of this peptide. Increased Abeta42 levels with 27-OHC are associated with increased levels of beta-amyloid precursor protein (APP) as well as beta-secretase (BACE1), the enzyme that cleaves APP to yield Abeta. Unchanged Abeta42 levels with 24-OHC are associated with increased levels of sAPPalpha, suggesting that 24-OHC favors the processing of APP to the non-amyloidogenic pathway. Interestingly, 24-OHC, but not 27-OHC, increases levels of the ATP-binding cassette transporters, ABCA1 and ABCG1, which regulate cholesterol transport within and between cells.

Conclusion: These results suggest that cholesterol metabolites are linked to Abeta42 production. 24-OHC may favor the non-amyloidogenic pathway and 27-OHC may enhance production of Abeta42 by upregulating APP and BACE1. Regulation of 24-OHC: 27-OHC ratio could be an important strategy in controlling Abeta42 levels in AD.

Figure 1

27-OHC, but not 24-OHC, increases levels of secreted Aβ42. While treatment with 5, 10 and 25 μM 24-OHC did not alter Aβ42 levels (a), treatment with 5, 10 and 25 μM 27-OHC significantly increased levels of Aβ42 compared to levels in medium of untreated cells (b). There was no difference in Aβ42 levels between untreated cells and cells treated with a mixture of 24-OHC + 27-OHC (c). **p < 0.01 (One way ANOVA followed by Dunnett's multiple comparison test).

Figure 2

27-OHC, but not 24-OHC, increases levels of APP and BACE1. Representative Western blots (a) and densitometric (b) analysis demonstrating increased levels of APP with 27-OHC. No changes were found in levels of APP with 24-OHC or 24-OHC+ 27-OHC treatment. BACE1 levels were unchanged with 24-OHC treatment but significantly increased with 27-OHC or a mixture of 24-OHC + 27-OHC. *p < 0.05, **p < 0.01 (One way ANOVA followed by Dunnett's multiple comparison test).

Figure 3

Immunofluorescence staining of APP and Aβ increases with 27-OHC. Immunostaining for APP and Aβ showed a decreased immunoreactivity to 6E10 antibody (green) in cells treated with 24-OHC compared treatment with 27-OHC. The immunoreactivity for 6E10 antibody in cells treated with a mixture of 24-OHC + 27-OHC is similar to that observed in control cells. DAPI (blue) was used as a nuclear counterstain. Bar 20 μm.

Figure 4

24-OHC increases processing of APP via the non-amyloidogenic pathway. Western blot (a) and densitometric analyses (b) demonstrating increased levels of sAPPα in medium of 24-OHC-treated cells. Treatment with 27-OHC or a mixture of 24-OHC + 27-OHC did not influence sAPPα levels. Levels of Aβ40 were not affected by treatment with 24-OHC, 27-OHC, or a mixture of 24-OHC + 27-OHC compared to levels in control cells (c). *p < 0.05 (One way ANOVA followed by Dunnett's multiple comparison test).

Figure 5

Treatment with 24-OHC, but not with 27-OHC, increased ABCA1 and ABCG1 levels. Representative Western blots (a) and densitometric analysis (b) showing increased levels ABCG1 and ABCA1 with 24-OHC treatment. Treatments with 27-OHC or with a mixture of 24-OHC + 27-OHC did not significantly change ABCA1 and ABCG1 levels. *p < 0.05 (One way ANOVA followed by Dunnett's multiple comparison test).