The Journal of Neuroscience, November 10, 2004 • 24(45):10191–10200 • 10191 Neurobiology of Disease Synaptic Targeting by Alzheimer’s-Related Amyloid ␤ Oligomers Pascale N. Lacor,1 Maria C. Buniel,1 Lei Chang,1 Sara J. Fernandez,1 Yuesong Gong,1 Kirsten L. Viola,1 Mary P. Lambert,1 Pauline T. Velasco,1 Eileen H. Bigio,2 Caleb E. Finch,3 Grant A. Krafft,4 and William L. Klein1 1Neurobiology and Physiology Department, Northwestern University, Evanston, Illinois 60208, 2Neuropathology Core, Northwestern Alzheimer’s Disease Center, Northwestern Feinberg School of Medicine, Chicago, Illinois 60611, 3Andrus Gerontology Center, University of Southern California, Los Angeles, California 90089, and 4Acumen Pharmaceuticals, Glenview, Illinois 60025 The cognitive hallmark of early Alzheimer’s disease (AD) is an extraordinary inability to form new memories. For many years, this dementia was attributed to nerve-cell death induced by deposits of fibrillar amyloid ␤ (A␤). A newer hypothesis has emerged, however, in which early memory loss is considered a synapse failure caused by soluble A␤ oligomers. Such oligomers rapidly block long-term potentiation, a classic experimental paradigm for synaptic plasticity, and they are strikingly elevated in AD brain tissue and transgenic- mouseADmodels.ThecurrentworkcharacterizesthemannerinwhichA␤oligomersattackneurons.Antibodiesraisedagainstsynthetic oligomers applied to AD brain sections were found to give diffuse stain around neuronal cell bodies, suggestive of a dendritic pattern, whereas soluble brain extracts showed robust AD-dependent reactivity in dot immunoblots. Antigens in unfractionated AD extracts attached with specificity to cultured rat hippocampal neurons, binding within dendritic arbors at discrete puncta. Crude fractionation showed ligand size to be between 10 and 100 kDa. Synthetic A␤ oligomers of the same size gave identical punctate binding, which was highly selective for particular neurons. Image analysis by confocal double-label immunofluorescence established that Ͼ90% of the punctate oligomer binding sites colocalized with the synaptic marker PSD-95 (postsynaptic density protein 95). Synaptic binding was accompanied by ectopic induction of Arc, a synaptic immediate-early gene, the overexpression of which has been linked to dysfunctional learning. Results suggest the hypothesis that targeting and functional disruption of particular synapses by A␤ oligomers may provide a molecular basis for the specific loss of memory function in early AD. Key words: ADDLs; amyloid; synapse; dendritic spines; immediate-early gene; arc Introduction including the poor correlation between dementia and amyloid Alzheimer’s disease (AD) is a progressive dementia that manifests plaque burden (Katzman et al., 1988). Studies of experimental in early stages primarily as a profound inability to form new vaccines with transgenic (tg) human amyloid protein precursor memories (Selkoe, 2002). The basis for this specificity is un- (hAPP) mice, which provide good models of early AD by devel- known, but evidence favors involvement of neurotoxins derived oping age-dependent plaques and memory dysfunction (Dodart from the self-associating amyloid ␤ peptide (A␤). A␤ generates et al., 2002; Kotilinek et al., 2002), have been particularly illustra- the fibrils of the hallmark amyloid plaques of AD, its production tive. When injected with monoclonal antibodies against A␤, is elevated by disease-associated mutations and risk factors, and these mice respond in ways unanticipated by the amyloid cas- fibrils made in vitro kill cultured brain neurons (Klein, 2004). The cade: (1) vaccinated mice show reversal of memory loss, with original amyloid cascade hypothesis, developed from these core recovery evident in 24 hr, and (2) cognitive benefits of vaccina- findings, thus attributed AD memory loss to neuron death caused tion accrue despite no decrease in plaque levels. Such findings are by fibrillar A␤ (Hardy and Higgins, 1992). inconsistent with a mechanism for memory loss dependent on Despite its intuitive appeal and strong experimental support, neuron death caused by amyloid fibrils. this hypothesis has proven inconsistent with key observations, A new version of the amyloid cascade hypothesis proposes an alternative mechanism for memory loss based on the impact of Received March 12, 2004; revised Sept. 30, 2004; accepted Sept. 30, 2004. small, soluble A␤ oligomers (Klein et al., 2001; Hardy and Selkoe, ThisworkwassupportedbygrantsfromtheIllinoisDepartmentofPublicHealth(P.N.L.);theAlzheimer’sDisease 2002). Its two salient new features are that (1) early memory loss Research Fund; National Institutes of Health–National Institute on Aging Grants RO1-AG18877, RO1-AG22547, and stems from synapse failure before neuron death and (2) synapse RO1-AG11385 (W.L.K.), RO3-AG022237 (P.N.L.), and P30AG13854 (Northwestern Alzheimer’s Disease Center); the ␤ Boothroyd,FeigerandFrenchFoundations;andtheInstitutefortheStudyofAging.WearegratefultoPierceCainfor failure derives from actions of A oligomers rather than fibrils. assistance with immunoblotting and William Russin of the Biological Imaging Facility of Northwestern University. This hypothesis emerged from experiments showing that A␤ oli- CorrespondenceshouldbeaddressedtoWilliamL.Klein,NorthwesternUniversity,NeurobiologyandPhysiology gomers rapidly inhibit long-term potentiation (LTP), a classic Department, O. T. Hogan Biological Sciences, 5-110, 2205 Tech Drive, Evanston, IL 60208. E-mail: experimental paradigm for memory and synaptic plasticity [email protected]. DOI:10.1523/JNEUROSCI.3432-04.2004 (Lambert et al., 1998; Walsh et al., 2002; Wang et al., 2002). Copyright © 2004 Society for Neuroscience 0270-6474/04/2410191-10$15.00/0 Recent findings that oligomers are strikingly elevated in hAPP 10192 • J. Neurosci., November 10, 2004 • 24(45):10191–10200 Lacor et al. • Basis for Synapse Failure in AD transgenic mice AD models (Chang et al., 2003) and, most signif- transferred to a 10–40% sucrose gradient. Free-floating 40-␮m-thick icantly, in AD brain (Gong et al., 2003) give the hypothesis sub- serial sections were obtained from frontal cortex and kept in cryopro- stantive clinical support. tectant at 4°C until immunolabeling. Sections were rinsed with TBS, Considerable interest is focused now on the mechanism by pretreated with 2% sodium m-periodate in TBS for 20 min, and perme- which oligomers interact with neurons (Caughey and Lansbury, abilized with 0.25% Triton X-100 in TBS (TBST). Aspecific immunore- activity (IR) was blocked with 5% horse serum in TBST for 40 min and 2003). Two prominent possibilities are that oligomers insert into 1% nonfat dry milk in TBST for 30 min. Sections were subsequently membrane bilayers to create cytotoxic pores, a relatively nonspe- incubated with M94 (1:1000) overnight at 4°C and Alexa Fluor 488 anti- cific mechanism, or alternatively, that oligomers bind to partic- rabbit IgG (1:500) for 90 min at RT. Serial sections were stained with ular membrane targets as highly specific ligands. Here, oligomer– 0.5% thioflavine-S in 50% ethanol. Confocal images were collected on neuron association has been examined using oligomers extracted the Leica confocal microscope as described above with z-series scans of 1 from AD brain or made in vitro. Results show that oligomers bind ␮m intervals. Similar sections were labeled with M94 antibody and sec- to neuron surfaces in small punctate clusters that colocalize al- ondary antibody–ABC complex and diaminobenzidine. Sections were most exclusively with a subpopulation of synaptic terminals. counterstained with hematoxylin. Control sections with the primary or Binding is accompanied by ectopic induction of Arc, a synaptic secondary antibodies omitted were negative. immediate-early gene the overexpression of which has been Immunoblot. Cells were lysed in 1 vol PBS–protease inhibitor mixture solution and 1 vol of 2ϫ loading buffer, pH 6.8 (80 mM Tris-HCl, 16.7% linked to dysfunctional learning. We hypothesize that targeting ␤ ␤ glycerol, 1.67% SDS, 1.67% -mercaptoethanol) and sonicated briefly. and functional disruption of particular synapses by A oligomers Proteins were separated on 4–20% Tris-glycine gels (Bio-Rad, Hercules, could underlie the specific loss of memory function in early AD. CA) at 100 V and transferred to nitrocellulose membrane at 100 V for 1 hr at 4°C in transfer buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 20% Materials and Methods v/v methanol). Blots were blocked with 5% nonfat dry milk in 10 mM A␤-derived diffusible ligands and fractionation.A␤1–42 peptide (California Tris-buffered saline containing 0.1% Tween 20, pH 7.5, for 2 hr. Blots Peptide Research, Napa, CA) or biotin-A␤1–42 peptide (Recombinant Pep- were incubated overnight at 4°C with anti-Arc antibody (1:250) and 2 hr tide, Athens, GA) was used to prepare synthetic A␤-derived diffusible li- with HRP-conjugated IgG (1:100,000). Membranes were developed with gands (ADDLs) or biotinylated ADDLs according to published protocols SuperSignal West Femto chemiluminescence kit (Pierce Biotechnology, (Lambert et al., 2001; Klein, 2002). Molecular weight (MW) fractionation of Rockford, IL) and then washed, blocked, and reblotted with anti- oligomeric species was obtained using Centricon YM-100 and YM-10 con- cyclophilin B antibody (1:40,000) used as a control for protein loading. centrators (Millipore, Bedford, MA), used according to manufacturer’s in- Proteins were visualized and quantified using the Kodak IS440CF Image structions.