Tournefolic acid B attenuates amyloid β protein-mediated toxicity by abrogating the calcium overload in mitochondria and retarding the caspase 8-truncated Bid-cytochrome c pathway in rat cortical neurons Chih-Wen Chi a , Yun-Lian Lin b , Ying-Hsiu Wang b , Chieh-Fu Chen a , Chuen-Neu Wang b, ⁎ , Young-Ji Shiao b,c, ⁎ a Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, ROC b National Research Institute of Chinese Medicine, Taipei, Taiwan, ROC c Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan, ROC Received 2 November 2007; received in revised form 29 January 2008; accepted 13 February 2008 Available online 29 February 2008 Abstract The effect of tournefolic acid B (TAB) on amyloid β protein-mediated neurotoxicity and the underlying mechanisms were investigated. Amyloid β protein 25–35 elicited neuronal death as determined by calcein/ethidium homodimer-1 staining. 10 μM amyloid β protein 25–35 caused cell death at a level of 41.5±3.8% by MTT reduction. 50 μM TAB attenuated the amyloid β protein 25–35-induced cell death by 49.7±11.1%. TAB also abrogated amyloid β protein-induced activation of caspases 8 and 9 by about 50–60%. Furthermore, TAB significantly diminished the amyloid β protein 25–35-induced elevation of calcium level in mitochondria, whereas it did not affect the calcium level in cytosol or endoplasmic reticulum. TAB markedly retarded the amyloid β protein-mediated release of cytochrome c from mitochondria. Amyloid β protein 25–35 elevated mitochondrial truncated BH3 interacting domain death agonist (tBid) and decreased the level of B-cell leukemia/lymphoma-2α (Bcl-2α) in mitochondria. Moreover, amyloid β protein induced a slight up-regulation of Bcl-2 agonist killer 1 (Bak) in cytosol. 50 μM TAB decreased the amyloid β protein-induced elevation of mitochondrial tBid and the level of Bak, whereas it did not affect the amyloid β protein-mediated decrease in mitochondrial Bcl-2α. Caspase 8 inhibitor significantly inhibited the amyloid β protein-mediated increase in mitochondrial tBid and the release of cytochrome c. Therefore, TAB blocked the overload of calcium in mitochondria and impaired the amyloid β protein-mediated activation of the caspase 8–tBid–cytochrome c pathway, thereby conferring its neuroprotective effects on amyloid β protein-mediated neurotoxicity. © 2008 Elsevier B.V. All rights reserved. Keywords: Tournefolic acid B; Amyloid β protein; Calcium; Caspase 8; Bid; Cytochrome c 1. Introduction Amyloid β protein is the major protein component of senile plaque, which is a hallmark of Alzheimer's disease and has been suggested to play an important role in the pathogenesis of Alzheimer's disease (Selkoe, 1990; Smith, 1998). Amyloid β protein is derived proteolytically from amyloid precursor protein (Buxbaum and Greengard, 1996; Selkoe, 1996). Abundant evidence has demonstrated that amyloid β protein is a cause of neuronal death and neuritic changes (Loo et al., 1993; Maurice et al., 1996; Estus et al., 1997; Mattson et al., 1998). Several different mechanisms have been suggested to be involved in amyloid β protein-induced neurotoxicity, which may be attri- butable to disturbing calcium homeostasis and accumulating reactive oxygen species (Maurice et al., 1996; MacManus et al., 2000). Reactive oxygen species can compromise membrane integrity by provoking membrane damage, which increases the permeability of ions, including calcium. Available online at www.sciencedirect.com European Journal of Pharmacology 586 (2008) 35 – 43 www.elsevier.com/locate/ejphar ⁎ Corresponding authors. Shiao is to be contacted at National Research Institute of Chinese Medicine, NO. 155-1. Sec. 2, LiNung ST., Peitou, Taipei, Taiwan, ROC. Tel.: +886 2 2820 1999x4171; fax: +886 2 28264266. Wang, National Research Institute of Chinese Medicine, NO. 155-1. Sec. 2, LiNung ST., Peitou, Taipei, Taiwan, ROC. Tel.: +886 2 2820 1999x4163; fax: +886 2 28264266. E-mail addresses: cnwang@nricm.edu.tw (C.-N. Wang), yshiao@nricm.edu.tw (Y.-J. Shiao). 0014-2999/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.02.058