Amyloid beta 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 opti mal activity. To test its pathophysiological changes in a phosphatidylinosi tol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheime r's disease (AD), amyloid beta proteins (A betas), on the Cl--ATPase activi ty were examined using primary cultured rat hippocampal neurons. Amyloid be ta 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 ele vated intracellular Cl- concentrations ([Cl-](i)), A beta 25-35 being the m ost potent. Addition of inositol or 8-Br-cyclic GMP completely reversed the se A beta -induced changes. The recoveries in enzyme activity were attenuat ed by an inhibitor of PI 4-kinase, 10 muM wortmannin or 20 mum quercetin, b ut not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 muM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the A beta -treated cells as compared with each control. In the A beta -ex posed culture, but not in control, stimulation by 10 tm glutamate for 10 mi n significantly increased fragmentation of DNA and decreased cell viability . Addition of inositol or 8-Br-cyclic GMP prevented the effect of A beta -t reatment on the neurotoxicity of glutamate. Thus, A betas reduce neuronal C l--ATPase activity, resulting in an increase in [Cl-](i) probably by loweri ng PI4P levels, and this may reflect a pre-apoptotic condition in early pat hophysiological profiles of AD.