Chloride-dependency of amyloid β protein-induced enhancement of glutamate neurotoxicity in cultured rat hippocampal neurons

In our previous studies, pathophysiological concentrations of amyloid-beta (Aβ) proteins increased intracellular Cl− concentration ([Cl−]i) and enhanced glutamate neurotoxicity in primary cultured neurons, suggesting Cl−-dependent changes in glutamate signaling. To test this possibility, we examined the effects of isethionate-replaced low Cl− medium on the Aβ-induced enhancement of glutamate neurotoxicity in the primary cultured rat hippocampal neurons. In a normal Cl− (135 mM) medium, treatment with 10 nM Aβ25–35 for 2 days increased neuronal [Cl−]i to a level three times higher than that of control as assayed using a Cl−-sensitive fluorescent dye, while in a low Cl− (16 mM) medium such an Aβ25–35-induced increase in [Cl−]i was not observed. The Aβ treatment aggravated glutamate neurotoxicity in a normal Cl− medium as measured by mitochondrial reducing activity and lactate dehydrogenase (LDH) release, while in a low Cl− medium the Aβ treatment did not enhance glutamate toxicity. Upon such Aβ plus glutamate treatment under a normal Cl− condition, activated anti-apoptotic molecule Akt (Akt-pS473) level monitored by Western blot significantly decreased to 74% of control. Under a low Cl− condition, a resting Akt-pS473 level was higher than that under a normal Cl− condition and did not significantly change upon Aβ plus glutamate treatment. Tyrosine phosphorylation levels of 110 and 60 kDa proteins (pp110 and pp60) increased upon Aβ plus glutamate treatment under a normal Cl−, but not low Cl−, condition. These findings indicated that Aβ-induced enhancement of glutamate neurotoxicity is Cl−-dependent. Chloride-sensitive Akt pathway and tyrosine phosphorylation of proteins (pp110 and pp60) may be involved in this process.