Amyloid β proteins inhibit Cl-ATPase activity in cultured rat hippocampal neurons

Authors


Address correspondence and reprint requests to Chiyoko Inagaki, Department of Pharmacology, Kansai Medical University, Fumizono-cho 10–15, Moriguchi-City, Osaka 570–8506, Japan. E-mail: inagaki@takii.kmu.ac.jp

Abstract

Cl-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid β proteins (Aβs), on the Cl-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid β proteins (1–40, 1–42 and 25–35) concentration-dependently (1–100 nm) and time-dependently (from 1 h to 6 day) decreased Cl-ATPase activity and elevated intracellular Cl concentrations ([Cl]i), Aβ25–35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Aβ-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 µm wortmannin or 20 µm quercetin, but not by a PI 3-kinase inhibitor, 50 nm wortmannin or 10 µm LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Aβ-treated cells as compared with each control. In the Aβ-exposed culture, but not in control, stimulation by 10 µm glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Aβ-treatment on the neurotoxicity of glutamate. Thus, Aβs reduce neuronal Cl-ATPase activity, resulting in an increase in [Cl]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.

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