O R I G I N A L R E S E A R C H Neurosteroids block the increase in intracellular calcium level induced by Alzheimer’s β-amyloid protein in long-term cultured rat hippocampal neurons Midori Kato-Negishi1 Masahiro Kawahara2 1Department of Developmental Morphology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183- 8526, Japan; 2Department of Analytical Chemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-cho, Nobeoka-shi, Miyazaki 882-8508, Japan Correspondence: Masahiro Kawahara Department of Analytical Chemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-cho, Nobeoka-shi, Miyazaki, 882-8508, Japan Tel +81 982 23 5706 Fax +81 982 23 5708 Email kawamasa@phoenix.ac.jp Abstract: The neurotoxicity of β-amyloid protein (AβP) is implicated in the etiology of Alzheimer’s disease. We previously have demonstrated that AβP forms Ca2+-permeable pores on neuronal membranes, causes a marked increase in intracellular calcium level, and leads to neuronal death. Here, we investigated in detail the features of AβP-induced changes in intracellular Ca2+ level in primary cultured rat hippocampal neurons using a multisite Ca2+- imaging system with fura-2 as a ﬂuorescent probe. Only a small fraction of short-term cultured hippocampal neurons (ca 1 week in vitro) exhibited changes in intracellular Ca2+ level after AβP exposure. However, AβP caused an acute increase in intracellular Ca2+ level in long-term cultured neurons (ca 1 month in vitro). The responses to AβP were highly heterogeneous, and immunohistochemical analysis using an antibody to AβP revealed that AβP is deposited on some but not all neurons. Considering that the disruption of Ca2+ homeostasis is the primary event in AβP neurotoxicity, substances that protect neurons from an AβP-induced intracellular Ca2+ level increase may be candidates as therapeutic drugs for Alzheimer’s disease. In line with the search for such protective substances, we found that the preadministration of neuros- teroids including dehydroepiandrosterone, dehydroepiandrosterone sulfate, and pregnenolone signiﬁcantly inhibits the increase in intracellular calcium level induced by AβP. Our results suggest the possible signiﬁcance of neurosteroids, whose levels are reduced in the elderly, in preventing AβP neurotoxicity. Keywords: neurotoxicity, pore, calcium homeostasis, channel, aging Introduction Alzheimer’s disease (AD) is a senile type of dementia affecting many of the elderly. AD is pathologically characterized by selective neuronal loss, and the presence of numerous extracellular deposits termed senile plaques and intraneuronal neuroﬁbril- lary tangles (NFTs) in the patient’s brain (Selkoe 1991). The principal constituent of senile plaques is the β-amyloid protein (AβP), which is a peptide composed of 38–43 amino acids and a proteolytic product of a large precursor protein (amyloid precursor protein; APP). Genetic studies of early-onset cases of familial AD indicated that APP mutations and AβP metabolism are associated with the disease (Goate et al 1991). Yankner and colleagues found that the segment comprising the ﬁrst 40 amino acid residues of AβP, termed AβP[1–40], has neurotoxic effects on primary cultured neurons of the rat hippocampus (Yankner et al 1990). These lines of evidence support the idea that AβP and its neurotoxicity might be causal agents of neuronal death in AD patients (Small et al 2001). Although the molecular mechanism underlying AβP neurotoxicity is not yet fully understood, there are a growing number of studies suggesting that the disruption of Ca2+ Neuropsychiatric Disease and Treatment 2008:4(1) 209–218 © 2008 Kato-Negishi and Kawahara, publisher and licensee Dove Medical Press Ltd.This is an Open Ac- cess article which permits unrestricted noncommercial use, provided the original work is properly cited. 209