Effects of cholesterol and its 24S-OH and 25-OH oxysterols on choline acetyltransferase-positive neurons in brain slices

Abstract

In Alzheimer's disease (AD) neurons expressing the enzyme choline acetyltransferase (ChAT) degenerate and a loss of cholinergic activity directly correlates with cognitive decline. Recent studies have suggested that cholesterol plays a role in AD. The aim of the present study was to explore if cholesterol and its oxysterols, 24S-hydroxycholesterol (24S-OH Chol) and 25-hydroxycholesterol (25-OH Chol), affect ChAT-positive neurons in organotypic brain slices of the basal nucleus of Meynert (nBM). We showed that slices expressed approximately 140 ChAT-positive neurons/slice after 2 weeks when incubated with nerve growth factor (NGF). This number markedly decreased when incubated without NGF to approximately 20 neurons/slice. Cholesterol and 24S-OH Chol delayed this decrease in ChAT-positive neurons. In contrast, 25-OH Chol induced a decline in ChAT-positive neurons in 2-week-old slices within 4 days. The effects of cholesterol and its oxysterols were exhibited in a dose- and time-dependent way. Our results show that cholesterol and 24S-OH Chol delays the decrease in ChAT-positive neurons, while 25-OH Chol rapidly decreases ChAT expression, suggesting differential mechanisms on ChAT expression in cholinergic nBM neurons.

Fig. 1

Organotypic brain slices of the nBM were cultured with (B, E) or without (A, D) 10 ng/ml NGF or with 2 μg/ml cholesterol (C, F). After 2 weeks slices were fixed and cholinergic neurons were immunohistochemically stained against ChAT (A–F). Higher magnifications of the corresponding slices are shown in D–F. Scale bar = 350 μm (A–C), 70 μm (D–F).

Fig. 2

Effects of cholesterol and 24S-OH Chol on ChAT in axotomized cholinergic brain slices (assay 1). Brain slices of the nBM were cultured without (−; ■) or with 10 ng/ml NGF (●) or with 0.02, 0.2, or 2 μg/ml cholesterol (□) (A, B) or 24S-OH Chol (▲) (C, D). After 2 weeks the slices were fixed and immunohistochemically stained against ChAT. The number of ChAT-positive neurons was counted in whole slices. Statistical analysis was performed by one-way ANOVA with a Fisher PLSD post hoc test. ***p < 0.001; *p < 0.05; NS = not significant.

Fig. 3

Effects of 25-OH Chol on ChAT expression in 2-week nBM brain slices (assay 2). Slices were incubated for 2 weeks with 10 ng/ml NGF, then for 3 days without NGF and then with (NGF, filled circles) or without (−) NGF, or 0.02, 0.2, 2 μg/ml 25-OH Chol (filled triangles). After 4 days brain slices were fixed and stained for ChAT. The number of ChAT-positive neurons was counted in whole slices. Statistical analysis was performed by one-way ANOVA with a Fisher PLSD post hoc test. ***p < 0.001; **p < 0.01; NS = not significant.