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, 115 (9), 2556-63

Alzheimer Disease Beta-Amyloid Activity Mimics Cholesterol Oxidase

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Alzheimer Disease Beta-Amyloid Activity Mimics Cholesterol Oxidase

Luigi Puglielli et al. J Clin Invest.

Erratum in

  • J Clin Invest. 2006 Oct;116(10):2828

Abstract

The abnormal accumulation of amyloid beta-peptide (Abeta) in the form of senile (or amyloid) plaques is one of the main characteristics of Alzheimer disease (AD). Both cholesterol and Cu2+ have been implicated in AD pathogenesis and plaque formation. Abeta binds Cu2+ with very high affinity, forming a redox-active complex that catalyzes H2O2 production from O2 and cholesterol. Here we show that Abeta:Cu2+ complexes oxidize cholesterol selectively at the C-3 hydroxyl group, catalytically producing 4-cholesten-3-one and therefore mimicking the activity of cholesterol oxidase, which is implicated in cardiovascular disease. Abeta toxicity in neuronal cultures correlated with this activity, which was inhibited by Cu2+ chelators including clioquinol. Cell death induced by staurosporine or H2O2 did not elevate 4-cholesten-3-one levels. Brain tissue from AD subjects had 98% more 4-cholesten-3-one than tissue from age-matched control subjects. We observed a similar increase in the brains of Tg2576 transgenic mice compared with nontransgenic littermates; the increase was inhibited by in vivo treatment with clioquinol, which suggests that brain Abeta accumulation elevates 4-cholesten-3-one levels in AD. Cu2+-mediated oxidation of cholesterol may be a pathogenic mechanism common to atherosclerosis and AD.

Figures

Figure 1
Figure 1
Cholesterol oxidase activity of Aβ:Cu2+. (A) Chemical structure of cholesterol showing oxidation of the hydroxyl group at position C-3 by cholesterol oxidase activity. (B and C) The migrations of nonoxidized cholesterol and 4-cholesten-3-one standards are shown. (B) Oxidation of cholesterol (50 μM) after incubation with BSA/Cu2+ (1 μM:2 μM), Aβ42 (1 μM), Cu2+ (2 μM), Aβ42:Cu2+ complex (1 μM:2 μM), or bacterial cholesterol oxidase (BCO; 5 IU). (C) Effect of Cu2+ chelation (with 1 mM TETA) on 4-cholesten-3-one generation by the Aβ42:Cu2+ complex; conditions as in B. Data are representative of at least 3 experiments. (D) Generation of 4-cholesten-3-one on TLC of Aβ40 and Aβ42 complexed with Cu2+ (1 μM:2 μM), and inhibition by the Cu2+ chelator CQ (100 μM). Bars show mean ± SD. Significance was calculated by 2-tailed Student’s t test of CQ effect. *P < 0.05. RU, relative units. (E) Total ion current mass chromatograms obtained by repetitive scanning GC-MS analysis of the underivatized compound and methyloxime derivative of the sample eluted from the TLC spot were superimposed for comparison with pure standard of 4-cholesten-3-one. The retention indices of each compound are denoted in MU. (F) Electron ionization (70 electron volts) mass spectrum of a compound identified as 4-cholesten-3-one by TLC from extracts of AD brain tissue eluted from the TLC plate: underivatized compound (top), 4-cholesten-3-one standard (middle), methyloxime derivative of the TLC extract (bottom; syn-isomer only shown) confirming the expected shift in masses of the molecular and fragment ions.
Figure 2
Figure 2
Catalytic oxidation of cell-free and cellular cholesterol by Aβ:Cu2+. (A) Production of 4-cholesten-3-one by Aβ42:Cu2+ (200 nM:400 nM) upon incubation for 60 minutes with increasing concentrations of nonoxidized cholesterol. Experiments were performed in triplicate. (B) Lineweaver-Burk transformation of the data shown in A. (C) Free cholesterol and (D) 4-cholesten-3-one in primary neurons after incubation for 2 days with Aβ42 (200 nM) and Aβ42:Cu2+ (200 nM:400 nM) with or without TETA (50 μM). Data are mean ± SD of 4 experiments. *P < 0.05.
Figure 3
Figure 3
Aβ:Cu2+-induced cholesterol oxidation correlates with toxicity in neuronal cultures. Hippocampal primary neurons were incubated with Aβ42:Cu2+ (200 nM:400 nM) with or without TETA (50 μM) for the indicated times. (A) Effect on 4-cholesten-3-one generation. (B) Effect on LDH release in the conditioned media. *P < 0.05. (C) Effect on the cleavage of full-length PARP (f.l. PARP; 116 kDa) into 85-kDa apoptosis-related (PARP-85) isoforms of PARP. Data are mean ± SD of 3 experiments.
Figure 4
Figure 4
Modified forms of Aβ with diminished toxicity form less 4-cholesten-3-one in neuronal cultures. Hippocampal primary neurons were incubated with Aβ42 variants (200 nM) plus Cu2+ (400 nM) for 3 days. (A) Effect on 4-cholesten-3-one generation. (B) Effect on LDH release in the conditioned media. **P < 0.001 vs. control; #P < 0.001 vs. Aβ42; ##P < 0.01 vs. Aβ1–42. (C) Association of 4-cholesten-3-one generated in the culture with LDH release. Baseline values of LDL (37.7 U/l) were subtracted for the purposes of illustration. Data are mean ± SEM of 3 experiments.
Figure 5
Figure 5
4-Cholesten-3-one is found in the brains of AD patients and AD transgenic mice. 4-cholesten-3-one (A and C) and nonoxidized cholesterol (B and D) levels in postmortem brain samples. (A and B) The inferior parietal lobe, matched for postmortem interval, of YC (age 32.0 ± 11.3 years, 4 males), PD (age 78.2 ± 8.5 years, 3 males), AC (age 78.5 ± 6.9 years, 2 males), and AD subjects (age 78.5 ± 7.5 years, 4 males). P value shown is for AD vs. AC subjects. (C and D) Tg2576 mice and non-Tg littermates (17 months old, 4 females per group).
Figure 6
Figure 6
Treatment of APP transgenic mice with CQ decreased brain 4-cholesten-3-one levels. (A) Effect on 4-cholesten-3-one generation. Data are mean ± SEM of n = 6 transgenic mice per group. (B) Association of total Aβ levels extracted from the pellet phase of brain homogenates with 4-cholesten-3-one levels.

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