A silver lining for 24-hydroxycholesterol in Alzheimer's disease: The involvement of the neuroprotective enzyme sirtuin 1

Abstract

It is now established that cholesterol oxidation products (oxysterols) are involved in several events underlying Alzheimer's disease (AD) pathogenesis. Of note, certain oxysterols cause neuron dysfunction and degeneration but, recently, some of them have been shown also to have neuroprotective effects. The present study, which aimed to understand the potential effects of 24-hydroxycholesterol (24-OH) against the intraneuronal accumulation of hyperphosphorylated tau protein, stressed these latter effects. A beneficial effect of 24-OH was demonstrated in SK-N-BE neuroblastoma cells, and is due to its ability to modulate the deacetylase sirtuin 1 (SIRT1), which contributes to preventing the neurotoxic accumulation of the hyperphosphorylated tau protein. Unlike 24-OH, 7-ketocholesterol (7-K) did not modulate the SIRT1-dependent neuroprotective pathway. To confirm the neuroprotective role of 24-OH, in vivo experiments were run on mice that express human tau without spontaneously developing tau pathology (hTau mice), by means of the intracerebroventricular injection of 24-OH. 24-OH, unlike 7-K, was found to completely prevent the hyperphosphorylation of tau induced by amyloid β monomers. These data highlight the importance of preventing the loss of 24-OH in the brain, and of maintaining high levels of the enzyme SIRT1, in order to counteract neurodegeneration.

Keywords: 24-hydroxycholesterol; Alzheimer's disease; Oxysterols; Sirtuin 1; Tau.

Fig. 1

Effect of 24-OH on SIRT1 expression and synthesis in SK-N-BE cells. (A) Gene expression was quantified in cells treated with 24-OH. Data, normalized to β2-microglobulin, are expressed as mean values ± SD of 4 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs control. (B) SIRT1 protein levels were analyzed in cells treated with 24-OH. The blot shown is representative of 3 experiments. The histograms represent mean values ± SD of 3 experiments. SIRT1 densitometric measurements were normalized against the corresponding α-tubulin levels and expressed as percentage of control value. *P < 0.05 vs control. (C) After cell treatment with 24-OH, SIRT1 protein levels were detected by confocal laser microscopy. The images are representative of 3 experiments.

Fig. 2

Effect of 7-K on SIRT1 expression and synthesis in SK-N-BE cells. (A) Gene expression was quantified in cells treated with 7-K. Data, normalized to β2-microglobulin, are expressed as mean values ± SD of 4 experiments. (B) SIRT1 protein levels were analyzed in cells treated with 7-K. The blot shown is representative of 3 experiments. The histograms represent mean values ± SD of 3 experiments. SIRT1 densitometric measurements were normalized against the corresponding α-tubulin levels and expressed as percentage of control value. (C) After cell treatment with 7-K, SIRT1 protein levels were detected by confocal laser microscopy. The images are representative of 3 experiments.

Fig. 3

Effect of 24-OH on the levels of acetylated, phosphorylated, and total tau in SK-N-BE cells. (A) Acetyl-tau, phospho-tau, and total tau levels were analyzed in cells treated with 24-OH. Each blot shown is representative of 3 experiments. The histograms represent mean values ± SD of 3 experiments. Densitometric measurements were normalized against the corresponding α-tubulin levels and expressed as percentage of control value. *P < 0.05 and ***P < 0.001 vs control. (B) Phospho-tau/tau ratio derived from densitometric measurements. *P < 0.05 and **P < 0.01 vs control.

Fig. 4

Effect of 7-K on the levels of acetylated, phosphorylated, and total tau in SK-N-BE cells. (A) Acetyl-tau, phospho-tau, and total tau levels were analyzed in cells treated with 7-K. Each blot shown is representative of 3 experiments. The histograms represent mean values ± SD of 3 experiments. Densitometric measurements were normalized against the corresponding α-tubulin levels and expressed as percentage of control value. (B) Phospho-tau/tau ratio derived from densitometric measurements.

Fig. 5

Effect of 24-OH and 7-K on ROS production and modulation of SIRT1 and phospho-tau levels by 24-OH in SK-N-BE cells. (A) Cells were treated with 24-OH or 7-K. The intracellular generation of ROS was monitored by DHE fluorescence staining. (B) The levels of SIRT1 and (C) phospho-tau were observed in cells incubated with 24-OH. Some cells were incubated with apocynin or with rotenone, and then treated with 24-OH. ROS, SIRT1, and phospho-tau levels were detected by confocal laser microscopy. The images are representative of 3 experiments. (D) Phospho-tau levels were analyzed in cells transfected with a specific SIRT1 siRNA and then incubated with 24-OH for 15 h. The blot is representative of 3 experiments and the histograms represent mean values ± SD of 3 experiments. Phospho-tau densitometric measurements were normalized against the corresponding α-tubulin levels and expressed as percentage of control value. ** p < 0.01 vs control; ## p < 0.01 vs 24-OH.

Fig. 6

24-OH protects against phosphorylated and total tau accumulation induced by Aβ_1–42 in mouse brain. hTau mice were ICV injected with 24-OH or 7-K for 4 days, and then with Aβ_1–42 or saline (control) for 3 h. Representative blots of brain extracts, using SIRT1, AT8 or Tau5. Densitometric measurements were normalized against the corresponding β-actin levels and expressed mean values ± SD, n = 5 each group. **p < 0.01 and ***p < 0.001 vs control; #p < 0.05 and ### p < 0.001 vs Aβ_1–42.