Up-regulation of β-amyloidogenesis in neuron-like human cells by both 24- and 27-hydroxycholesterol: protective effect of N-acetyl-cysteine

Abstract

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK-N-BE human neuroblastoma cells with patho-physiologically relevant amounts of 27-OH and 24-OH showed that both oxysterols induce a net synthesis of Aβ1-42 by up-regulating expression levels of amyloid precursor protein and β-secretase, as well as the β-secretase activity. Interestingly, cell pretreatment with N-acetyl-cysteine (NAC) fully prevented the enhancement of β-amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β-amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols-induced Aβ toxic peptide accumulation in the brain.

Keywords: 24-hydroxycholesterol; 27-hydroxycholesterol; Alzheimer's disease; BACE1; N-acetyl-cysteine; amyloid β.

Figure 1

Effect of 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) on the expression and synthesis of the amyloid precursor protein (APP). (A) Gene expression was quantified by real-time RT–PCR in differentiated SK-N-BE cells treated for times up to 12 h with 1 μm 27-OH or 24-OH. Untreated cells were taken as control. Data, normalized to β2-microglobulin, are expressed as mean values ± SD of four different experiments. **P < 0.01, and ***P < 0.001 versus control group. (B) APP protein levels were analyzed by Western blotting in differentiated SK-N-BE cells treated up to 48 h with 1 μm 27-OH or 24-OH. Untreated cells were taken as control. APP densitometric measurements were normalized against the corresponding β actin levels. The experiments were conducted in triplicate. *P < 0.05, and **P < 0.01 versus control group.

Figure 2

Effect of 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) on the expression and synthesis of β-secretase (BACE1). (A) Gene expression was quantified by real-time RT–PCR in differentiated SK-N-BE cells treated for times up to 12 h with 1 μm 27-OH or 24-OH. Untreated cells were taken as control. Data, normalized to β2-microglobulin, are expressed as mean values ± SD of four different experiments. *P < 0.05, and ***P < 0.001 versus control group. (B) BACE1 protein levels were analyzed by Western blotting in SK-N-BE cells treated up to 48 h with 1 μm 27-OH or 24-OH. Untreated cells were taken as control. BACE1 densitometric measurements were normalized against the corresponding β actin levels. The experiments were conducted in triplicate. **P < 0.01 versus control group.

Figure 3

Effect of 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) on the expression and synthesis of the γ-secretase subunity presenilin 1 (PS1). (A) Gene expression was quantified by real-time RT–PCR in SK-N-BE cells treated for times up to 12 h with 1 μm 27-OH or 24-OH. Untreated cells were taken as control. Data, normalized to β2-microglobulin, are expressed as mean values ± SD of four different experiments. *P < 0.05 versus control group. (B) The C-terminal fragment (CTF) of PS1 (CTF-PS1) levels were analyzed by Western blotting in SK-N-BE cells treated up to 48 h with 1 μm 27-OH or 24-OH. Untreated cells were taken as control. CTF-PS1 densitometric measurements were normalized against the corresponding β actin levels. The experiments were conducted in triplicate. *P < 0.05, and **P < 0.01 versus control group.

Figure 4

Effect of 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) on the expression and synthesis of α-secretase (ADAM10). (A) Gene expression was quantified by real-time RT–PCR in differentiated SK-N-BE cells treated for times up to 12 h with 1 μm 27-OH or 24-OH. Untreated cells were taken as control. Data, normalized to β2-microglobulin, are expressed as mean values ± SD of four different experiments. **P < 0.01, and ***P < 0.001 versus control group. (B) ADAM10 protein levels were analyzed by Western blotting in SK-N-BE cells treated up to 48 h with 1 μm 27-OH or 24-OH. Untreated cells were taken as control. ADAM10 densitometric measurements were normalized against the corresponding β actin levels. The experiments were conducted in triplicate. ***P < 0.001 versus control group.

Figure 5

27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) induce Aβ_1-42 production by up-regulating BACE1 and γ-secretase enzymatic activities in SK-N-BE cells. Differentiated SK-N-BE cells were incubated up to 48 h with 27-OH or 24-OH. Untreated cells were used as control. BACE1 activity (A) and γ-secretase activity (B) were measured by fluorogenic assay using the secretase-specific substrate conjugated to the fluorescent reporter molecules. Data were expressed as percentage change versus activity of control cells. Data are means ± SD of three experiments. *P < 0.05 versus control group. (C) Differentiated SK-N-BE cells were incubated for 24 h with 27-OH or 24-OH. Untreated cells were used as control. Aβ_1-42 intracellular concentration was quantified by enzyme-linked immunoassay (ELISA). Data are means ± SD of three experiments. ***P < 0.001 versus control group.

Figure 6

Up-regulation of BACE1 and γ-secretase and Aβ_1-42 over-production are prevented by cell pretreatment with N-acetyl-cysteine (NAC). Differentiated SK-N-BE cells were incubated for 24 h with 27-hydroxycholesterol (27-OH) or 24-hydroxycholesterol (24-OH). Some cell aliquots were also pre-incubated for 1 h with 100 μm NAC. Untreated cells were used as control. (A) The C-terminal fragment (CTF) of PS1 (CTF-PS1) and BACE1 protein levels were analyzed by Western blotting. CTF-PS1 and BACE1 densitometric measurements were normalized against the corresponding β actin levels. The experiments were conducted in triplicate. *P < 0.05, and **P < 0.01 versus control group; #P < 0.05, and ##P < 0.01 versus oxysterol groups. (B) Aβ_1-42 intracellular concentration was quantified by enzyme-linked immunoassay (ELISA). Histograms represent the mean values ± SD of three experiments. ***P < 0.001 versus control group, and ^###P < 0.001 versus 27-OH or 24-OH.