Amyloid β-protein potentiates tunicamycin-induced neuronal death in organotypic hippocampal slice cultures

We have assessed amyloid β protein (Aβ)–induced neurotoxicity, with and without added tunicamycin (TM), an inhibitor of N-glycosylation in the endoplasmic reticulum (ER), in rat organotypic hippocampal slice cultures (OHCs). In the rat OHCs cultured for 3 weeks, there was little neurotoxicity after treatment with Aβ25-35 (25 μM) alone for 48 h. However, with TM alone, concentration-dependent neuronal death was observed at concentrations between 20 and 80 μg/mL. When amyloid-β protein was combined with tunicamycin (Aβ+TM), cell death was more acute than with TM alone. Western blot analysis revealed that calpain activity and the active forms of caspase-12 and caspase-3 was increased after exposure to Aβ+TM as compared with exposure to TM alone. In contrast, the levels of glucose regulated protein (GRP)94, GRP78 and C/EBP homologous protein (CHOP) were not changed in the presence of Aβ. Aβ potentiation of TM neurotoxicity was reversibly blocked by S-allyl-l-cysteine (SAC), an organosulfur compound purified from aged garlic extract, and the L-type calcium channel blocker, nifedipine, in a restricted neuronal area of the OHCs. Simultaneously applied SAC also reversed the increases in calpain activity and the active forms of caspase-12 and caspase-3 by Aβ+TM with no change in the increased levels of GRP94, GRP78 and CHOP. These data indicate that Aβ facilitates the calpain–caspase-12–caspase-3 pathway, thus potentiating TM-induced neuronal death in the hippocampus.