CHARACTERIZATION OF NEURONAL DYSTROPHY INDUCED BY FIBRILLAR AMYLOID L : IMPLICATIONS FOR ALZHEIMER’S DISEASE E. A. GRACE, C. A. RABINER and J. BUSCIGLIO Ã Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA AbstractöAmyloid deposition, neuronal dystrophy and synaptic loss are characteristic pathological features of Alzheimer’s disease (AD). We have used cortical neuronal cultures to assess the dystrophic e¡ect of ¢brillar amyloid L (AL) and its relationship with neurotoxicity and synaptic loss. Treatment with ¢brillar AL led to the development of neuritic dystrophy in the majority of the neurons present in the culture. Morphometric analysis and viability assays showed that neuronal dystrophy appeared signi¢cantly earlier and at lower AL concentrations than neurotoxicity, suggesting that both e¡ects are generated independently by di¡erent cellular mechanisms. The development of dystrophic features required AL ¢bril formation and did not depend on the presence of the RHDS adhesive domain in the sequence of AL. Finally, a dramatic reduction in the density of synaptophysin immunoreactivity was closely associated with dystrophic changes in viable neurons. These results suggest that aberrant plastic changes and loss of synaptic integrity induced by ¢brillar AL may play a signi¢cant role in the development of AD pathology. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: neurotoxicity, cell adhesion, cortical cultures, amyloid plaques, neuronal plasticity, synaptic density. Alzheimer’s Disease (AD) is a progressive neurological disorder of the elderly characterized by memory loss and dementia. The hallmark of AD neuropathology is the presence of neuro¢brillary tangles and amyloid plaques in the brain of a¥icted patients (Selkoe, 1991). Neuro- ¢brillary tangles are intra-neuronal bundles of paired helical ¢laments composed primarily of hyper-phosphor- ylated tau protein. Amyloid plaques are extracellular lesions composed of a core of ¢brillar amyloid L (AL) surrounded by dystrophic neurites and reactive glial cells. In addition, several structural and biochemical abnor- malities are observed in the cerebral cortex including neuronal loss, reduced synaptic density and depletion of neurotransmitters (Selkoe, 1991). Genetic evidence supports a role for ¢brillar AL in the development of AD pathology (Selkoe, 2001). Mutations in the amyloid precursor protein (APP) and presenilins that cause familial AD promote AL ¢bril formation by increasing the cellular production of either all forms of AL, speci¢cally AL42, or by increasing the assembly of AL proto¢brils (Haass and Steiner, 2001). The patholog- ical relevance of AL deposition in AD is further sug- gested by studies indicating that ¢brillar AL can be neurotoxic both in vitro and in vivo (Yankner et al., 1990; Pike et al., 1991; Mattson et al., 1992; Geula et al., 1998). In the AD brain, aberrant neuronal sprouting can be observed surrounding amyloid plaques and in areas of synaptic loss in the hippocampal formation and neocor- tex (Geddes et al., 1986; Masliah et al., 1991). The dys- trophic pathology characteristic of AD seems to be associated with AL deposition and is not found in other neurodegenerative conditions (Benzing et al., 1993). In culture, AL acts as a permissive substrate for neuronal growth (Koo et al., 1993; Sabo et al., 1995), suggesting that AL may modulate neuritic outgrowth. One possibility is that the cell adhesion domain RHDS, spanning residues 5^8 of AL, may interact with integrin- like receptors on the cell surface and activate down- stream signal transduction pathways (Ghiso et al., 1992; Sabo et al., 1995; Saporito-Irwin and Van Nostrand, 1995). In this regard, several studies indicate that AL deposition induces neuronal dystrophy in culture (Pike et al., 1992) and in transgenic models (Irizarry et al., 1997; Phinney et al., 1999), but the precise relation- ship between AL-induced neuronal dystrophy and neuro- nal degeneration has not yet been characterized. To address this issue, we analyzed the time course of AL-induced neuritic dystrophy and neuronal cell death in rat cortical neuronal cultures treated with ¢brillar AL. Our results provide evidence for a dynamic process in which AL ¢brils cause progressive neuronal dystrophy and synaptic alterations starting at earlier stages of AL treatment, followed by a gradual decline in neuronal via- 265 *Corresponding author. Tel.: +1-860-679-3578; fax: +1-860-679- 8766. E-mail address: busciglio@nso1.uchc.edu (J. Busciglio). Abbreviations: AL, amyloid L ; AD, Alzheimer’s disease; APP, amy- loid precursor protein; DMEM, Dulbecco’s modi¢ed Eagle’s medium; PBS, phosphate-bu¡ered saline. NSC 5684 12-8-02 Cyaan Magenta Geel Zwart www.neuroscience-ibro.com Neuroscience Vol. 114, No. 1, pp. 265^273, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(02)00241-5 0306-4522 / 02 $22.00+0.00