Diazoxide and cyclosporin A protect primary cholinergic neurons against beta-amyloid (1-42)-induced cytotoxicity.

Objective: Activation of mitochondrial (MitoK(ATP)) channels was found to protect against anoxic and chemical stress in brain. This present study sought to investigate the ability of diazoxide and cyclosporin A to antagonize cytotoxicity induced by beta-amyloid peptide (A-beta(1-42)) in cultured rat primary basal forebrain cholinergic neurons. Methods: Cytotoxicity was induced by A-beta(1-42) (2 mu M) in the presence of either diazoxide (500 mu M), a selective opener of the mitochondrial ATP-sensitive potassium channel (MitoK(ATP)), or cyclosporin A (20 mu M), an inhibitor of the mitochondrial permeability transition pore (MTP), or the combination of both the reagents. We determined cell morphology and cell viability using MTT assay and expression levels of antiapoptotic protein (Bcl-2), pro-apoptotic proteins (Bax, cytochrome C, caspase-3 and cleaved caspase-3) using Western blotting at 24 hours and 72 hours. Results: Cell viability decreased markedly after exposure to A-beta(1-42) for 72 hours with a decrease in the expression of Bcl-2 protein and cytochrome C and an increase in the caspase-3 and cleaved caspase-3 levels. Both diazoxide and cyclosporin A exerted significant protective effects on cell viability by ameliorating the decrease in Bcl-2 and the increase in cytochrome c and caspase-3 activity induced by A-beta(1-42). The combination of both the reagents had a greater protective effect than either one alone. Conclusions: The present research demonstrates that activation of MitoK(ATP) channels independently or in combination with inhibitors of the MTP can elicit a protective effect against primary cholinergic neuron cytotoxicity induced by A-beta(1-42). These findings suggest new mitochondrial targets for the development of therapeutic agents against A-beta-induced cytotoxicity.