ORIGINAL PAPER Mutual Stimulation of Beta-Amyloid Fibrillogenesis by Clioquinol and Divalent Metals Silvia Bolognin Æ Paolo Zatta Æ Denise Drago Æ Pier Paolo Parnigotto Æ Fernanda Ricchelli Æ Giuseppe Tognon Received: 10 April 2008 / Accepted: 30 July 2008 / Published online: 19 August 2008 Ó Humana Press 2008 Abstract As reported by some authors, clioquinol (CQ), a 8-hydroxyquinoline derivative, has produced very encouraging results in the treatment of Alzheimer’s disease (AD). Its biological effects are most likely ascribed to complexation of specific metal ions, such as copper (II) and zinc (II), critically associated with b-amyloid (Ab) aggre- gation/fibrillogenesis and degeneration processes in the brain. The present study was aimed at assessing the in vitro effects of CQ on the aggregation/fibrillogenesis properties of human Ab either alone or complexed with Cu 2? and Zn 2? . Surprisingly, our data indicated that CQ promoted rather than inhibited the formation of Ab fibrillar aggre- gates when added metal ions were present. To understand whether the latter effects were related to the peptide amino acid sequence, we also investigated the aggregational profile of rat Ab, which differs from the human homolo- gous for three amino acidic substitutions. Such a sequence alteration drastically reduced the tendency of the peptide to undergo spontaneous aggregation/fibrillization. In the presence of CQ and metals, however, also rat Ab showed a strong propensity to generate fibrillar aggregates. In agreement with the pro-aggregation effects observed in solution, studies with neuroblastoma cells demonstrated an impairment of cell functioning only in the presence of CQ ? Ab–metals. Based on the present findings, the literature data on the potential effectiveness of CQ-based chelation therapy in AD should be re-interpreted. Keywords Clioquinol Á Metal ions Á b-Amyloid Á Aggregation/fibrillogenesis Introduction Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. The disease is characterized by loss of neurons in the cerebral cortex and by the presence of extraneuronal senile plaques whose core is constituted by a peptide mixture of 39–43 residues called b-amyloid (Ab). Ab is derived from a larger, type I transmembrane protein called b-amyloid precursor protein (APP). During APP processing by b and c-secretases, this precursor pro- tein generates Ab. Outside the cell Ab monomers aggregate into clumps called oligomers which accumulate and form the deposits known as mature senile plaques. It has been proposed that Ab aggregation follows a sequence which includes the formation of soluble, low molecular weight (LMW) oligomers which are present prior to fibril formation and include dimers, tetramers and dodecameric forms of Ab (Walsh et al. 2005; Lesne ´ et al. 2006). The aggregation process is extremely dynamic and oligomers associate rapidly to form higher-order aggregates. The nature of the Ab aggregates is a crucial issue. It is well known that Ab is toxic in vitro to cultured neurons (Yanker 1996); nevertheless, whether the oligomeric or the fibrillated form (plaques) of Ab is the toxic species remains an object of debate (Drouet et al. 2000). Until the last decade, the amyloid cascade hypothesis suggested that the insoluble and fibrillar form of Ab might S. Bolognin Á P. Zatta Á D. Drago Á F. Ricchelli (&) Á G. Tognon Department of Biology, Padova ‘‘Metalloproteins’’ Unit, CNR-Institute of Biomedical Technologies, University of Padova, Via Ugo Bassi 58B, 35121 Padova, Italy e-mail: rchielli@mail.bio.unipd.it P. P. Parnigotto Department of Pharmaceutical Sciences, University of Padova, Padova, Italy Neuromol Med (2008) 10:322–332 DOI 10.1007/s12017-008-8046-x