Cholesterol secoaldehyde, an ozonation product of cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons

Aldehydic products from ozonation of cholesterol and peroxidation of phospholipids have been shown to accelerate aggregation of amyloid-β (Aβ) in vitro. Here, we show that 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco), an ozonation product of cholesterol, induces Aβ aggregation, generation of reactive oxygen species (ROS), and cytotoxicity in murine GT1-7 hypothalamic neurons. The formation of Aβ aggregates in situ was dose-dependent at ChSeco concentrations ranging from 1t o 20µM. The increase in insoluble Aβ aggregates at increasing concentrations of ChSeco was accompanied by a decrease in soluble Aβ as evidenced by Western blot analysis. The formation of ROS in neuronal cells was found to be dose- and time-dependent with the magnitude being higher at 20 µM compared to 10 µM ChSeco or untreated controls. The increase in ROS was associated with depletion of GSH. The cytotoxicity induced by ChSeco involved changes in phosphatidylserine translocation, DNA fragmentation, and caspase 3/7 activity that are characteristic of apoptosis. Pretreatment of neuronal cells with Trolox, a water-soluble analog of α-tocopherol offered partial, but significant protection against ChSeco-induced cell death, whereas, N-acetyl-L-cysteine (NAC) completely prevented the cytotoxic effects of ChSeco. NAC and Trolox were without any effects on ChSeco-induced Aβ aggregation. Fibrillogenesis inhibitors, which inhibited A β aggregation, did not inhibit cell death induced by ChSeco, implying that ROS generation, and not Aβ aggregation, plays a major role in the observed cytotoxicity. However, since Alzheimer's and other neurodegenerative diseases are slow and progressive, the formation of Aβ aggregates in vivo by ChSeco may have long-term pathological consequences. Abbreviations: AD, Alzheimer's diseases; BSA, bovine serum albumin; ChSeco, cholesterol secoaldehyde; CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; CM-DCF, 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein; GST, glutathione-S-transferase; KRH, Krebs-Ringer-HEPES; HRP, horseradish peroxidase; MTS, 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NAC, N-acetyl-L-cysteine; PMS, phenazine methosulfate; PS, phosphatidylserine; ROS, reactive oxygen species; and TBST, Tris-buffered saline with 0.1% (w/v) Tween 20.