Alzheimer’s amyloid b-peptide (1–42) induces cell death in human neuroblastoma via bax/bcl-2 ratio increase: An intriguing role for methionine 35 q M.E. Clementi a , M. Pezzotti a , F. Orsini a , B. Sampaolese a , D. Mezzogori b , C. Grassi b , B. Giardina a , F. Misiti c, * a CNR-ICRM, Institute of ‘‘Chimica del Riconoscimento Molecolare’’, c/o Institute of Biochemistry and Clinical Biochemistry, Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome, Italy b Institute of Physiology, Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome, Italy c Department of Health and Motor Sciences, University of Cassino, Viale Bonomi, 03043 Cassino, (FR), Italy Received 24 January 2006 Available online 3 February 2006 Abstract The b amyloid (Ab), the major protein component of brain senile plaques in Alzheimer’s disease, is known to be directly responsible for the production of free radicals toxic to brain tissue and the redox state of Met-35 residue seems to play a particular and critical role in peptide’s neurotoxic actions. In this study, we investigated, in human neuroblastoma cells (IMR-32), the relationship between the oxi- dative state of methionine, and both neurotoxic and pro-apoptotic actions induced by Ab-peptide, comparing the effects of native pep- tide, in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 (Ab(1–42) 35Met-ox ), as well as an Ab-derivative with Met-35 substituted with norleucine (Ab(1–42) 35Nle ). The obtained results show that Ab induces a time-depen- dent decrease in cell viability; Ab(1–42) 35Met-ox was significantly less potent, though inducing a remarkable decrease in cell viability com- pared to control. On the contrary, no toxic effects were observed after treatment with Ab(1–42) 35Nle .Ab-peptide as well as the amyloid modified peptide with oxidized Met-35 induced the pro-apoptotic gene bax over-expression after 24 h, whereas Ab(1–42) 35Nle had no effect. Conversely, bcl-2, an anti-apoptotic gene, became highly down-regulated by Ab peptide treatment, in contrast to that evidenced by the Ab(1–42) 35Met-ox peptide. Finally, Ab caused an increase in caspase-3 activity to be higher with respect to that shown by Ab(1– 42) 35Met-ox while Ab(1–42) 35Nle had no effect. These results support the hypothesis that Ab-induced neurotoxicity occurs via bax over- expression, bcl-2 down-regulation, and caspase-3 activation, first indicating that methionine 35 redox state may alter this cell death pathway. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Ab(1–42) peptide; Methionine; Apoptosis; bax; bcl-2; Caspase Alzheimer’s disease (AD) is a brain pathology character- ized by the presence of senile plaques in several regions of the central nervous system (CNS), especially in those areas where neurodegeneration occurs [1]. A major protein com- ponent of the plaques is the amyloid peptide (Ab), a 39–43 amino acid peptide derived from a larger transmembrane protein, amyloid precursor protein (APP). Although con- troversial, it is established that Ab-induced neurotoxicity occurs through the induction of apoptotic pathways [2–4]. The prevailing amyloid hypothesis of AD holds that b amyloid becomes toxic when it adopts a fibrillar conforma- tion and that fibrillar Ab deposition in senile plaques (SP) causes neuronal degeneration. However, recent studies have shown that soluble forms of b amyloid exhibited 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.01.137 q Abbreviations: Ab, amyloid b-peptide (Ab(1–42)); Ab(1–42) 35Nle , Ab(1–42)35Met fi Nle; Ab(1–42) 35Met-ox ,Ab(1–42)35Met fi Met35 sulf- oxide. * Corresponding author. Fax: +39063053598. E-mail address: f.misiti@unicas.it (F. Misiti). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 342 (2006) 206–213 BBRC