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

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.