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

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 n m ) 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 µ m wortmannin or 20 µ m quercetin, but not by a PI 3‐kinase inhibitor, 50 n m wortmannin or 10 µ m 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 µ m 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.