Leptin reduces the accumulation of Abeta and phosphorylated tau induced by 27-hydroxycholesterol in rabbit organotypic slices

Abstract

Accumulation of amyloid-beta (Abeta) peptide and deposition of hyperphosphorylated tau protein are two major pathological hallmarks of Alzheimer's disease (AD). We have shown that cholesterol-enriched diets and its metabolite 27-hydroxycholesterol (27-OHC) increase Abeta and phosphorylated tau levels. However, the mechanisms by which cholesterol and 27-OHC regulate Abeta production and tau phosphorylation remain unclear. Leptin, an adipocytokine involved in cell survival and in learning, has been demonstrated to regulate Abeta production and tau hyperphosphorylation in transgenic mice for AD. However, the involvement of leptin signaling in cholesterol and cholesterol metabolites-induced Abeta accumulation and tau hyperphosphorylation are yet to be examined. In this study, we determined the effect of high cholesterol diet and 27-OHC on leptin expression levels and the extent to which leptin treatment affects 27-OHC-induced AD-like pathology. Our results show that feeding rabbits a 2% cholesterol-enriched diet for 12 weeks reduces the levels of leptin by approximately 80% and incubating organotypic slices from adult rabbit hippocampus with 27-OHC reduced leptin levels by approximately 30%. 27-OHC induces a 1.5-fold increase in Abeta (40) and a 3-fold increase in Abeta (42) and in phosphorylated tau. Treatment with leptin reversed the 27-OHC-induced increase in Abeta and phosphorylated tau by decreasing the levels of BACE-1 and GSK-3beta respectively. Our results suggest that cholesterol-enriched diets and cholesterol metabolites induce AD-like pathology by altering leptin signaling. We propose that leptin administration may prevent the progression of sporadic forms of AD that are related to increased cholesterol and oxidized cholesterol metabolite levels.

Fig. 1

Feeding rabbits a 2% cholesterol-enriched diet for 12 weeks (n = 6) reduced leptin protein (a and b) and mRNA expression (c) levels in hippocampus compared to control animals (n = 6). Treatment of organotypic slices from rabbit hippocampus (30 sections per hippocampus per rabbit; n = 4 rabbits) with 27-OHC for 72 h also reduced leptin protein (d and e) and mRNA expression (f) levels. *p < 0.05; **p < 0.01 versus control.

Fig. 2

Treatment with 27-OHC increases Aβ₄₀ and Aβ₄₂ levels both in organotypic slices (a and b) and in media (c and d). Treatment with leptin markedly reduced Aβ₄₀ and Aβ₄₂ levels in organotypic slices (a and b) and in media (c and d). Incubation of organotypic slices with leptin alone reduced Aβ₄₀ and Aβ₄₂ levels both in organotypic slices (a and b) and Aβ₄₀ in media (c) compared to control slices. **p < 0.01; ***p < 0.001 versus control. †††p < 0.001 versus 27-OHC treatment. 15 sections from each of 4 rabbits were used for each treatment. (e) Hippocampal slices treated with 27-OHC (panel B) show intense immunoreactivity to 6E10, an antibody that detect Aβ (and AβPP), compared to control slices (panel A) that show lesser immunoreactivity to 6E10. The 6E10 antibody stains cell body as well as cell processes. Leptin treatment alone (panel C) or in combination with 27-OHC (panel D) greatly reduced the immunoreactivity to 6E10.

Fig. 3

Western blots (a) and densitometric analyses (b-d) demonstrate increased levels of AβPP and BACE-1 and decreased levels of sAβPPα following treatment of organotypic slices with 27-OHC. Treatment with leptin reduced the 27-OHC-induced increase in AβPP and BACE-1 and increased levels of sAβPPα. **p < 0.05 versus control. †p < 0.05, ††p < 0.01, †††p < 0.001 versus 27-OHC treatment. 15 sections from each of 4 rabbits were used for each treatment.

Fig. 4

Western blots (a) and densitometric analysis (b and c) showing decreased levels of LRP-1 and IDE in organotypic slices treated with 27-OHC compared to control slices. Leptin treatment reversed the 27-OHC-induced decrease in LRP-1 and IDE levels. *p < 0.05 versus control and †p < 0.05 versus 27-OHC treatment. 15 sections from each of 4 rabbits were used for each treatment.

Fig. 5

Treatment with 27-OHC increases levels of phosphorylated tau in organotypic slices, as detected by PHF-1 antibody (a and b) and reduces levels of inactive phosphorylated Ser⁹ GSK-3β (c and d), potentially increasing tau phosphorylation. Leptin reverses the 27-OHC induced increase in tau phosphorylation by increasing the levels of Ser⁹ GSK-3β (a-d). *p < 0.05, **p < 0.01 versus control and †p < 0.05 versus 27-OHC treatment. 15 sections from each of 4 rabbits were used for each treatment.

Fig. 6

High cholesterol diets lead to increased levels of plasma cholesterol (hypercholesterolemia). Hypercholesterolemia reduces transport of leptin from the circulation into the brain and increases plasma levels of the cholesterol metabolite 27-OHC, thus allowing excess entry of 27-OHC into the brain and reduction of leptin expression in this organ. Low levels of leptin in the hippocampus trigger Aβ production by increasing AβPP levels, fostering AβPP processing by BACE-1, and reducing AβPP processing by the non-amyloidogenic α pathway. Increased levels of 27-OHC and reduced leptin expression in the brain also decrease the degradation and clearance of Aβ by reducing levels of IDE and LRP-1 respectively, thereby leading to the accumulation of this peptide. 27-OHC also increases the phosphorylation of tau (p-tau) by increasing the activity of GSK-3β. (+), increased; (−), decreased.