Integrins Mediate b-Amyloid-Induced Cell-Cycle Activation and Neuronal Death Giuseppina Frasca, 1 Viviana Carbonaro, 1,2 Sara Merlo, 1,3 Agata Copani, 4 and Maria Angela Sortino 1 * 1 Department of Experimental and Clinical Pharmacology, University of Catania, Catania, Italy 2 Neuropharmacology PhD program, University of Catania, Catania, Italy 3 DISCAFF, University of Piemonte Orientale, Novara, Italy 4 Department of Pharmaceutical Sciences, University of Catania, Catania, Italy Early intracellular events responsible for cell-cycle induction by b-amyloid (Ab) in neurons have not been identified yet. Extracellular signal–regulated kinases 1/2 (ERK1/2) have been identified in this pathway, and inhi- bition of ERK activity prevents cell-cycle activation and reduces neuronal death induced by Ab. To identify upstream events responsible for ERK activation, atten- tion has been focused on integrins. Treatment of SH- SY5Y cells, differentiated by long-term exposure to 10 lM retinoic acid with a neutralizing anti-a1-integrin anti- body significantly reduced Ab-induced neuronal death. However, cell-cycle analysis showed that treatment with anti-a1-integrin per se produced changes in the distribution of cell populations, thus hampering any effect on Ab-induced cell-cycle activation. 4-Amino-5- (4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide, an in- hibitor of src protein kinases that colocalizes with focal adhesion kinase (FAK) and is involved in integrin signal- ing, was effective in reducing activation of the cell cycle and preventing induction of neuronal death by Ab while inhibiting ERK1/2 phosphorylation. Similar results were obtained when FAK expression was down-regulated by siRNA silencing. The present study identifies a sequence of early events in the toxic effect of Ab in neuronal cultures that involves interaction with integ- rins, activation of FAK/src, enhanced phosphorylation of ERK1/2, and induction of the cell cycle, all leading to neuronal death. V V C 2007 Wiley-Liss, Inc. Key words: cell cycle; neuronal death; FAK; ERK Although neurons have been classically recognized as postmitotic cells, evidence has accumulated in recent years that injured neuronal cells have the ability to acti- vate the cell cycle. Such a phenomenon also has been described as occurring in the brains of patients with Alz- heimer’s disease (AD), strongly supporting a role for cell-cycle abnormalities in the pathogenesis of AD. Thus, reexpression of several cell-cycle-related proteins, such as cdc2, cyclin B1, cdk4, cyclin D, and p16, has been reported in sick as well as in vulnerable neurons in the brains of AD patients (Nagy et al., 1997; Vincent et al., 1997; Busser et al., 1998; McShea et al., 1999; Husseman et al., 2000). Similar reexpression of cell- cycle-related proteins has been also reported in experi- mental mouse models of AD (Herrup et al., 2004). A focused analysis of the reappearance of cell-cycle markers and their relationship to cell-cycle activation revealed that primary cultures of rat cortical neurons challenged with Ab (25–35) enter the cell cycle, as demon- strated by the appearance of a cell population in the S phase of the cycle. This phenomenon is accompanied by sequential and ordered expression of cell-cycle proteins such as phospho-Rb and cyclins and is thought to lead to neuronal apoptosis (Copani et al., 1999). Although the intracellular pathway of cell-cycle activation and neuronal death by Ab has been drawn, early transducing mechanisms operating in the induction of the cell cycle by Ab have not been clearly identified and described. To better understand the early mechanisms under- lying Ab-induced cell-cycle activation, we used a human neuroblastoma cell line, SH-SY5Y cells, differentiated by a 5-day exposure to retinoic acid. We previously showed that in this cell line (1) Ab (25–35) induces modifications in the distribution of cells in different phases of the cell cycle, an event that precedes neuronal apoptosis; and (2) the extracellular signal–regulated kinase (ERK) pathway is involved in this effect (Frasca et al., 2004). To analyze possible pathways upstream of ERK, we focused our attention on integrins that, among others, have been identified as potential mediators of the early response to Ab (Kowall et al., 1991; Boland et al., 1995; Sabo et al., 1995; Yan et al., 1996; Yaar et al., 1997). Binding to integrins resulted in the consecutive activation of tyro- Contract grant sponsor: University of Catania institutional research grant (to M.A.S.). *Correspondence to: Dr. Maria Angela Sortino, Department of Experi- mental and Clinical Pharmacology, University of Catania, Viale Andrea Doria 6, 95125 Catania Italy. E-mail: msortino@unict.it Received 26 January 2007; Revised 15 June 2007; Accepted 28 June 2007 Published online 7 September 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21487 Journal of Neuroscience Research 86:350–355 (2008) ' 2007 Wiley-Liss, Inc.