Previously, we found that amyloid b-protein (Ab)1-42 exhibits neurotoxicity, while Ab1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Ab1-40 against Ab1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Ab1-42 at concentrations higher than 2 lM, which was prevented by concurrent treatment with Ab1-40 in a dosedependent manner. However, metal chelators did not prevent Ab1-42-induced neuronal death. Circular dichroism spectroscopy showed that Ab1-40 inhibited the b-sheet transformation of Ab1-42. Thioflavin-T assay and electron microscopy analysis revealed that Ab1-40 inhibited the fibril formation of Ab1-42. In contrast, Ab1-16, Ab25-35, and Ab40-1 did not inhibit the fibril formation of Ab1-42 nor prevent Ab1-42-induced neuronal death. Ab1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Ab1-40. These results indicate that Ab1-40 protects neurons from Ab1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the b-sheet transformation and fibril formation of Ab1-42. Our data suggest a mechanism by which elevated Ab1-42/Ab1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.

Hypoxia is an important feature of many diseases such as malignant solid tumors, inflammatory diseases and cardiac ischemia. We herein focused on the development of a novel hypoxia-sensitive fluorescent probe, IQ-R, consisting of an indolequinone unit and a rhodol fluorophore. IQ-R has good solubility in water and longer wavelength for absorption and emission, which are favorable for cellular bioimaging. While the fluorescence of rhodol in the IQ-R conjugate was quenched by the function of intramolecular indolequinone unit, it was restored under hypoxic conditions through the enzymatic one-electron reduction of IQ-R by NADPH:cytochrome P450 reductase to release the nonconjugated free rhodol. When administered to A549 cells, IQ-R was activated and reduced by endogenous reductase preferentially under hypoxic conditions, thereby visualizing hypoxic cancer cells by robust fluorescence.

A characteristic feature of the reactivity of indolequinone derivatives, substituents of which can be removed by one-electron reduction under hypoxic conditions, was applied to the development of a new class of fluorescent probes for disease-relevant hypoxia. A reducing indolequinone parent molecule conjugated with fluorescent coumarin chromophores could suppress efficiently the fluorescence emission of the coumarin moieties by an intramolecular electron-transfer quenching mechanism and a conventional internal-filter effect. Under hypoxic conditions, however, the conjugate, denoted IQ-Cou, underwent a one-electron reduction triggered by X irradiation or the action of a reduction enzyme to release a fluorescent coumarin chromophore, whereupon an intense fluorescence emission with a maximum intensity at 420 nm was observed. The one-electron reduction of IQ-Cou was suppressed by molecular oxygen under aerobic conditions. IQ-Cou also showed intense fluorescence in a hypoxia-selective manner upon incubation with a cell lysate of the human fibrosarcoma cell line HT-1080. The IQ-Cou conjugate has several unique properties that are favorable for a fluorescent probe of hypoxia-specific imaging.

Biological reduction of fluorine-labeled indolequinone derivative (IQ-F) was characterized by (19)F NMR for quantitative molecular understanding. The chemical shift change in (19)F NMR allowed monitoring of the enzymatic reduction of IQ-F. Upon hypoxic treatment of IQ-F with NADPH:cytochrome P450 reductase, IQ-F was activated via catalytic one-electron reduction to release nonafluoro-tert-butyl alcohol (F-OH), while the formation of F-OH was significantly suppressed under aerobic conditions. Similar hypoxia-selective reduction of IQ-F occurred within A549 cells, which expresses NADPH:cytochrome P450 reductase. The kinetic analysis was also performed to propose a reaction mechanism. The molecular oxygen slightly prevents the binding of IQ-F to reductase, while the rate of net reaction was decreased due to oxidation of a semiquinone anion radical intermediate generated by one-electron reduction of IQ-F. The disappearance of IQ-F and appearance of F-OH were imaged by (19)F fast spin echo, thus visualizing the hypoxia-selective reduction of IQ-F by means of MR imaging.

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