Anti-ADDL antibodies differentially block oligomer binding to hippocampal neurons

Neurobiology of Aging 31 (2010) 189–202

Anti-ADDL antibodies differentially block oligomer binding to hippocampal neurons P.J. Shughrue a,∗ , P.J. Acton a,1 , R.S. Breese a,1 , W.-Q. Zhao a , E. Chen-Dodson a , R.W. Hepler b , A.L. Wolfe a , M. Matthews a , G.J. Heidecker b , J.G. Joyce b , S.A. Villarreal a , G.G. Kinney a b

a Department of Alzheimer’s Research, Merck Research Laboratories, West Point, PA 19486, USA Department of Vaccine and Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA

Received 10 September 2007; received in revised form 31 March 2008; accepted 2 April 2008 Available online 16 May 2008

Abstract A␤-derived diffusible ligands (ADDLs) are abundant in AD brain, bind to hippocampal neurons and induce deficits in rodent cognition. To further investigate ADDL binding to neurons and identify antibodies that block this association, a panel of anti-A␤ and anti-ADDL antibodies was characterized for their ability to immuno-detect neuronally bound ADDLs and attenuate the binding of ADDLs to neurons. The results showed that anti-A␤ and anti-ADDL antibodies were able to abate ADDLs binding to hippocampal neurons, but to different degrees. Quantitative assessment of binding showed that one antibody, ACU-954 was markedly more effective at blocking ADDL binding than other antibodies assessed. ACU-954 was also found to block ADDL binding to hippocampal slice cultures, attenuate the ADDL-induced loss of dendritic spines and detect “natural ADDLs” in human AD tissue. These results demonstrated that antibodies that bind to and block ADDL binding to neurons can be identified, although their efficacy is conformationally specific since it is not readily apparent or predictable based on the core linear epitope or affinity for monomeric A␤. © 2008 Published by Elsevier Inc. Keywords: Neurodegeneration; Alzheimer’s disease; Amyloid; Oligomers; Immunotherapy

1. Introduction Alzheimer’s disease (AD) is characterized by the progressive loss of cognitive function and eventual accumulation of amyloid ␤ (A␤) plaques in regions involved with learning and memory. Accumulating evidence now suggests that A␤-derived diffusible ligands (ADDLs) and not A␤ plaques, per se, play a critical role in the pathogenesis of AD (Gong et al., 2003; Kayed et al., 2003). ADDLs are small, soluble oligomers of A␤ that are abundant in AD, but not in normal brain (McLean et al., 1999; Gong et al., 2003; ∗ Corresponding author at: Department of Alzheimer’s Research, Merck Research Laboratories, Mail Stop, WP44K, West Point, PA 19486, USA. Tel.: +1 215 652 4816; fax: +1 215 652 2075. E-mail address: paul [email protected] (P.J. Shughrue). 1 These authors contributed equally to this paper.

0197-4580/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.neurobiolaging.2008.04.003

Lambert et al., 2007). In vitro studies have shown that naturally produced (from AD brain) or synthetic ADDLs, bind to a sub-population of cortical and hippocampal neurons (Gong et al., 2003; Klein et al., 2004; Lacor et al., 2004; Lambert et al., 2007) while little or no binding was detected with fibrillar or monomeric A␤ preparations (Lacor et al., 2004; Hepler et al., 2006). In rodents, the central administration of ADDLs induced the loss of synapses and deficits in rodent long-term potentiation (LTP) and memory formation (Walsh et al., 2002; Cleary et al., 2005; Klyubin et al., 2005). The effect of ADDLs on LTP was attenuated when ADDLs were co-administered with an anti-A␤ antibody (6E10 or 4G8) or administered to animals actively immunized with A␤ peptide (Rowan et al., 2004). In transgenic mouse models that over express human amyloid precursor protein (hAPP), age-associated cognitive deficits have been observed, with the onset of impairment

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associated with elevated ADDL levels (Westerman et al., 2002; Ashe, 2005; Lee et al., 2005; Lesne et al., 2006). Interestingly, when these same hAPP mice were treated with an anti-ADDL antibody, a significant improvement in cognition was observed without a concomitant decrease in plaque load (Lee et al., 2005). Together these findings indicate that ADDLs and not plaques are primarily responsible for cognitive impairment. Moreover, the data suggest that an antiADDL antibody may prove efficacious in the treatment of AD. To date, some antibodies that detect ADDLs by Western or dot blot have been described. These include antibodies that detect monomeric, ADDL and fibrillar forms of A␤ (e.g. 6E10 and 4G8; see Lambert et al., 1998; Dahlgren et al., 2002; Kayed et al., 2003; Klyubin et al., 2005; Ma et al., 2006; Lesne et al., 2006), ADDL preferring antibodies (e.g. NU-1, M71, M93, and M94; Lambert et al., 2001, 2007; Gong et al., 2003; Lacor et al., 2004) and a pan oligomer antibody (A11; Kayed et al., 2003). While these antibodies have advanced our understanding of ADDL formation and biological activity, it has been difficult to compare and contrast the efficacy of antibodies. To this end, we used immunocytochemistry and a quantitative cell-based binding assay to assess ADDL binding to neurons and the ability of a panel of anti-A␤ and anti-ADDL antibodies to block this association. Herein, we describe the identification of a highly effective antiADDL antibody that binds to and blocks ADDL binding to neurons.

2. Materials and methods 2.1. Preparation of ADDLs and bADDLs ADDLs were prepared using previously described methods (Lambert et al., 2001; Hepler et al., 2006). Briefly, synthetic A␤1–42 peptide (American Peptide, CA) was dissolved in hexafluoro-2-propanol (HFIP) at a concentration of 10 mg/ml, and incubated at room temperature (RT) for 1 h. The peptide solution was transferred to polypropylene microcentrifuge tubes (50 ␮l/tube), the HFIP removed with a Speedvac, and the resulting peptide films stored desiccated at −70 ◦ C. A 0.5 mg dried HFIP film was dissolved in 22 ␮l of anhydrous dimethyl sulfoxide (DMSO) with agitation for 10 min on a vortex mixer. Subsequently, 1 ml of cold Ham’s F12 media without phenol red (Biosource, CA) was added to the DMSO/peptide mixture. The tube was capped, inverted to insure complete mixing and incubated overnight at 4 ◦ C. The next morning samples were centrifuged for 10 min at 10,000 × g at 2–8 ◦ C. The supernatant was carefully transferred to a new tube and stored at 2–8 ◦ C until used. Biotinylated ADDLs (bADDLs) were prepared using the same method, but starting with 100% N-terminal biotinylated A␤1–42 peptide (American Peptide, CA). The ADDL and bADDL preparations were always used within 1–4 days after generation and stored at 4 ◦ C until use. A time-course analysis of preparations with Western blot and size exclu-

sion chromatography showed that there was no detectable difference during this short storage period. In the present manuscript, we define ADDLs as any soluble oligomer of A␤ consisting of 3–48-mer A␤ peptides. Due to the heterogeneity of A␤ aggregation states produced using this method (see Hepler et al., 2006), molar concentrations of unfractionated ADDL or bADDL preparations refer to the molar concentration of starting A␤1–42 peptide. Moreover, the term oligomers, ADDLs, globulomers, etc. have been inconsistently used to refer to overlapping forms of A␤ oligomeric states. 2.2. Anti-ADDL antibodies BALB/c mice were immunized with ADDLs (prepared at Northwestern University; see Lambert et al., 1998) and the lymphocytes isolated and fused with myeloma cells to generate hybridomas according to established protocols (Immuno-Precise Antibodies Ltd., Victoria, BC). Monoclonal antibodies were then produced and purified for use (QED Bioscience, CA). 2.3. Neuronal cultures Primary hippocampal cultures were prepared from frozen dissociated rat hippocampal cells from embryonic day 18 Sprague–Dawley rats (Cambrex Corp., MD) that were thawed and plated in 96-well plates (Corning, NY) or on 12well round cover glass inserts at a concentration of 20,000 or 60,000 cells/well or prepared fresh in our laboratory. The cells were maintained in media (Neurobasal without l-glutamine, supplemented with B27; Invitrogen, CA) for a period of 7–21 days in vitro (DIV) and then used for binding studies. B103 neuroblastoma cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) without phenol red (Invitrogen, CA), 10% fetal bovine serum (Hyclone, UT) and 1% PenStrep (Invitrogen, CA). Exponentially growing cells were dissociated and plated in 96-well plates (Corning, NY) at a concentration of 5000 cells/well. Twenty-four hours after plating, cells were used to assess ADDL and bADDL binding. CHO cells were grown in DMEM without phenol red (Invitrogen, CA), 10% fetal bovine serum (Hyclone, UT), 2 mM l-glutamine (Invitrogen, CA) and 1% Pen-Strep (Invitrogen, CA). Exponentially growing CHO cells were dissociated and plated in 96-well plates (Corning, NY) at a concentration of 5000 cells/well. Twenty-four hours after plating, cells were used to assess ADDL and bADDL binding. To generate hippocampal slice cultures, Long Evans rats (Taconic Farms, NY) were euthanized at postnatal day 12, the hippocampi removed and 300–400 ␮m slices were generated using a McIlwain tissue chopper (Campden Instruments, IN). The slices were transferred to a 0.4 ␮m membrane insert (Millipore, MA), placed in multi-well culture plates with modified minimum essential medium (MEM) (Invitrogen, CA), maintained for 48 h (see Muller et al., 2000) and then used for binding studies.

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2.4. Biophysical characterization Atomic force microscopy was performed as described previously (Hepler et al., 2006) using a MultiMode AFM (Digital Instruments/Veeco Metrology, Santa Barbara, CA) controlled by a NanoScope IIIa with NanoScope Extender electronics and Q-Control (nanoAnalytics, M¨unster, Germany). Size exclusion chromatography was performed on an Agilent (Wilmington, DE) 1100 series HPLC system equipped with a diode-array detector, a Wyatt Technology (Santa Barbara, CA) Optilab DSP interferometric refractometer, and a Wyatt DAWN EOS multiangle laser light scattering (MALLS) detector as described previously (see Hepler et al., 2006). 2.5. ADDL and bADDL binding The binding of ADDLs or bADDLs to neurons was detected using standard immunofluorescence procedures. Primary hippocampal neurons (14–21 DIV), B103 cells (plated for 24 h), CHO cells (plated for 24 h) or hippocampal slice cultures were incubated with a 5–25 ␮M ADDLs or bADDLs preparation (based on starting peptide concentration) for 1 h at 37 ◦ C and washed 3–4 times with warm culture media (see above) to remove unbound ADDLs or bADDLs. The cells were fixed with 4% paraformaldehyde [16% paraformaldehyde (Electron Microscopy Sciences, PA), diluted in phosphate-buffered saline without calcium, magnesium, or phenol red (PBS; Invitrogen, CA)], permeabilized (4% paraformaldehyde solution with 0.1% Triton-X 100; Sigma, MO) for 10 min, washed with PBS and then incubated for 1 h at 37 ◦ C with blocking buffer (PBS with 10% bovine serum albumin, BSA; Sigma, MO). At that step, the protocols for the detection of bound ADDLs and bADDLs diverge. To detect ADDL binding, cells were incubated overnight at 37 ◦ C with 6E10 (Signet Labs, MA; diluted 1:1000 in PBS containing 1% BSA) or one of a panel of anti-ADDL monoclonal antibodies (diluted 1:1000). To visualize cell processes, a polyclonal antiserum raised against total tau protein (Sigma, MO; 1:1000) was utilized. During the cell characterization studies, cells were also stained with anti-GABA (Sigma; A2052, 1:100), antiGAD 65 (Chemicon; AB5082, 1:100) or GFAP (Chemicon; AB5804, 1:100) antibodies. After incubation with antibody, cells were washed with PBS, incubated for 1 h at RT with Alexa 594-labeled streptavidin to visualize bADDLs and an Alexa 488-labeled anti-rabbit secondary (Invitrogen, CA; diluted 1:1000), washed in PBS and the binding observed using a microscope with fluorescence capabilities. For the detection of bADDL binding, cells were incubated overnight with a total tau antibody to detect processes (see above), washed and incubated for 1 h at RT with an Alexa 488labeled anti-rabbit secondary (see above) and an Alexa 594-labeled streptavidin (1:500 dilution; Invitrogen, CA). The cells were then washed and the binding visualized with a fluorescence microscope. The cell nuclei were labeled

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with 4 ,6-diamidino-2-phenylindole (DAPI) (1:1000 dilution; Invitrogen, CA) according to standard protocols. 2.6. Blockade of bADDL binding with anti-Aβ or anti-ADDL antibodies The ability of antibodies to block the binding of ADDLs or bADDLs to neuronal cultures was characterized using methods described above with some modifications. Antibodies [monoclonal or a non-relevant IgG (mouse IgG control; Jackson, PA)] were mixed with a 1–10 ␮M bADDLs preparation at a ratio of 1:1, 5:1, or 20:1 (bADDLs:antibody) and incubated for 1 h at 37 ◦ on a slow rotator (Macs Mix; Miltenyi Biotec, Auburn, CA). Subsequently, the bADDL/antibody mixture was added to cells for 1 h at 37 ◦ C. After incubation, cells were washed, fixed, permeabilized, blocked, and the bADDLs were visualized with fluorescent or brightfield methods. For brightfield, bADDL binding was detected with a standard ABC method (Vector Elite kit, Vector, CA). Briefly, the sections were washed in PBS (2× 10 min) and then Tris (pH 7.6, 1× 10 min) prior to the 10 min DAB reaction (40 mg 3,3 -diaminobenzidine, 80 mg Ni, 12 ␮l 30% H2 O2 ). The sections were washed in Tris, mounted on slides, dehydrated, and cover-slipped. For the blockade studies using 6E10 and ADDLs, ADDL binding was detected with a rabbit anti-ADDL antibody (M71, 1:10,000) and a goat anti-rabbit secondary antiserum (Jackson, PA, 1:500). 2.7. ADDL-induced synaptic drebrin loss To assess ADDL and bADDL-induced changes in synaptic spines and the protective function of anti-ADDL antibodies, we used drebrin staining as a marker of dendritic spines. Rat primary hippocampal neurons (21 DIV) were treated with ADDLs or bADDLs (0.5, 1.0 or 3 ␮M) for 24 h and drebrin visualized with a mouse (1:200; Stressgen, Ann Arbor, MI) or rabbit (1:200 Sigma–Aldrich, St. Louis, MO) anti-drebrin antibody. To evaluate the efficacy of anti-ADDL antibodies in protecting neurons from the ADDL-induced synaptotoxicity, ADDLs or bADDLs were pre-incubated with native or heat-denatured antibodies (ADDL:antibody ratio 1:1 or 10:1) for 1 h as described above. The changes in drebrin staining in synaptic spines were then quantified using the Image J 1.37 V program (Open Source, Public Domain). For bADDL binding and synaptic drebrin levels, appropriate thresholds were applied to the images across experimental conditions, and the mean gray levels and integrate intensities were measured. The values were then normalized against the number of neurons from each image plate and compared statistically among different treatments. To assess changes in spine numbers, the puncta of drebrin staining in spines were counted using the Particle Count function of Image J. Statistic analyses were performed by one-way ANOVA using the GraphPad Prism5 software. p-Values <0.05 were considered significant.

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2.8. Quantification of bADDL binding and blockade with anti-Aβ or anti-ADDL antibodies In order to quantitatively assess the degree of bADDL binding and the ability of anti-A␤ and anti-ADDL antibodies to abate this interaction, we developed a cell-based alkaline phosphatase assay. Antibodies or PBS were mixed at a 5:1 ratio (bADDL:antibody) with a 2.5–10 ␮M bADDLs preparation and incubated for 1 h at 37 ◦ C on a slow rotator (4 rpm). After pre-incubation, the bADDL/antibody preparations were added to primary cultures and incubated for 1 h at 37 ◦ C. The cells were washed, fixed with 4% paraformaldehyde (2× 10 min at RT), permeabilized with 4% paraformaldehyde/0.1% Triton X-100 (2× 10 min at RT), washed (6× PBS) and treated with 10% BSA in PBS for 1 h at 37 ◦ C. Alkaline phosphatase conjugated streptavidin (Invitrogen, CA; 1:1500 in 1% BSA) was added to cells for 1 h at RT. The cells were rinsed 6 times with PBS, the alkaline

phosphatase substrate (CDP Star with Sapphire-II; Applied Biosystems, CA) added to the cells and incubated for 30 min prior to determining the luminescence on a LJL Luminometer (Analyst AD, Biosystems).

3. Results 3.1. Characterization of ADDLs and biotin-labeled ADDLs The use of biotin-labeled ADDLs (bADDLs) provides a simple, non-biased method to detect bound ADDLs and directly evaluate the blockade of ADDL binding with antibodies of interest. Initial studies examined both the biophysical and functional properties of ADDLs and bADDLs (Fig. 1) as well as their ability to bind hippocampal neurons (Fig. 2). As shown by atomic force microscopy

Fig. 1. ADDL and bADDL preparations were evaluated and characterized with atomic force microscopy (AFM; A) and size exclusion chromatography (SEC; B). Note that ADDLs and bADDLs have a similar footprint by SEC (B), although the percentage of biotin used in the bADDL preparations clearly affects the percentage of high molecular weight species. Data from AFM shows that the size of oligomers generated with bADDLs is more homogeneous than ADDLs (A). In addition, ADDLs and bADDLs were assessed for their ability to attenuate drebrin staining, a marker of dendritic spines on hippocampal neurons (C–G). The results show that both ADDLs and bADDLs significantly (** p < 0.01, *** p < 0.001) reduce the number of dendritic spines.

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Fig. 2. The ability of ADDLs or bADDLs to bind to primary rat hippocampal neurons was assessed using immunocytochemistry. When primary hippocampal cells were incubated with a 5 ␮M ADDLs (A) or bADDLs (B) preparation and binding visualized (ADDLs with monoclonal ACU-954; bADDLs with streptavidin), a sub-population of hippocampal neurons with a punctate pattern of binding (red staining) was observed. Interestingly, other hippocampal neurons (processes visualized with total tau, green) and glia (nuclei without processes) in these cultures had very little or no binding. A co-localization study revealed that bADDLs, bound to neurons (C), were detected with an anti-ADDL monoclonal antibody (D, ACU-954). Note the overlap of bADDL binding and anti-ADDL immunoreactivity (E, yellow), thus demonstrating that bADDLs can be used to visualize ADDL binding sites. The effect of age (DIV) on the binding of bADDLs to hippocampal neurons is shown in figures F–K. After 1 wk, little binding was seen on the neuronal processes (F–G), while the degree of binding is elevated at 2 wks (H–I) and most pronounced at 3 wks in culture (J–K). The scale bar for C–E is the same.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

(Fig. 1A) and size exclusion chromatography (Fig. 1B), both A␤1–42 and biotinylated-A␤1–42 peptide readily formed a heterogenous mixture of monomers and multimers, with high molecular weight species (≥12 mers) being the predominate oligomers. Importantly, these same preparations are found to consist largely of trimer and tetrameric species when evaluated by other methods (e.g., SDS-PAGE, see Bitan et al., 2005; Hepler et al., 2006), suggesting that the assignment of oligomer size for these ADDL and bADDL preparations is dependent on the method of evaluation. Further analysis indicated that the biotinylated A␤1–42 peptide appeared to produce more high molecular weight species (Fig. 1A and B), when compared with the standard ADDL preparation, and these oligomers were more homogeneous in size. Recent studies showed that oligomer binding alters synaptic structure and function (Lacor et al., 2007; Shankar et al., 2007).

To assess the ability of ADDLs and bADDLs to modulate neuronal structure, a dendritic spine protein, drebrin was evaluated in primary neurons (Fig. 1C–G). The results showed that both ADDLs (Fig. 1F) and bADDLs (Fig. 1G) significantly reduced the number of drebrin-containing dendritic spines on hippocampal neurons after 24 h of exposure (see Fig. 1C) and indicated that the subtle biophysical differences in ADDL and bADDL preparations did not markedly affect cell binding or subsequent downstream signaling events. It is worth noting that a 1 ␮m concentration of ADDLs was more efficacious at attenuating drebrin staining in neurons than the 3 ␮m concentration, although the reason for this is currently unknown. Since previous work had shown that high molecular weight species and not monomer bind to primary hippocampal neurons (Hepler et al., 2006), additional studies were conducted with ADDLs and bADDLs. As shown

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in Fig. 2A and B, both ADDLs and bADDLs bound to neuronal processes and the cell soma, with the pattern of binding being comparable and consistent with previous reports using ADDLs (Gong et al., 2003; Klein et al., 2004; Lacor et al., 2004). To further investigate the effect of biotin on antibody recognition of oligomers, several anti-ADDL antibodies were used to detect bound bADDLs. The results showed that bADDLs bind to a subset of neurons (Fig. 2C) and demonstrated that a number of different anti-ADDL antibodies (ACU-954; Fig. 2D) could detect bound bADDLs. The superimposition of bADDL binding and detection with an anti-ADDL antibody further revealed a marked overlap in staining (Fig. 2E). The present observations, data from Western blot studies (not shown) and other observations indicated that the biotin-label does not alter binding or recognition of oligomers by antibodies. During the course of these studies we also noted that the number of labeled neurons and degree of staining increased with age in culture (Fig. 1F–K), with specific staining first seen after 7 DIV and increasing until approximately day 21. This observation bolsters the belief that ADDLs bind to a specific site on neuronal membranes, a site that is cell specific and temporally regulated. The results of control studies (e.g. no ADDLs or no antibody and no bADDLs) further

demonstrated the specificity of ADDL and bADDL binding and detection with antibody. 3.2. Phenotypic characterization of ADDLs binding neurons It is intriguing that only a sub-population of the primary hippocampal neurons binds ADDLs in vitro. To elucidate the phenotype of cells that bind bADDLs, a series of co-localization studies were conducted with markers for glutamate and GABA. The results showed a high percentage of GABAergic (Fig. 3A) and glutamatergic (Fig. 3C) neurons bind bADDLs, although some GABAergic neurons and processes were clearly unlabeled (Fig. 3B). In contrast, the glial cells present in these mixed hippocampal cultures were unlabeled (Fig. 3D). 3.3. Blockade of bADDL binding with anti-ADDL or Aβ antibodies The ability of antibodies to block bADDL binding to hippocampal neurons in vitro was assessed with immunocytochemistry. As a first step, two anti-ADDL antibodies

Fig. 3. Co-localization studies showing bADDL binding to GABAergic (A–B) and glutamatergic (C) neurons using immunocytochemical methods. Note that some GABAergic neurons bind ADDLs (A), while others have unlabeled processes that extend out alone in culture and/or encircle labeled neurons (B). A high percentage of glutamate neurons also bind bADDLs (C), while the glial cells in these mixed hippocampal cultures are unlabeled (D). The scale bar is the same for images B–D.

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(ACU-954 and ACU-966), a putative pan anti-oligomer antibody (A11) and a commercially available anti-A␤ antibody (6E10) were evaluated for their ability to detect ADDLs bound to neurons. The results revealed that anti-ADDL (Fig. 4B) and anti-A␤ (Fig. 4C) antibodies detected the neuronal-bound ADDLs, while the antibody A11 and mouse IgG (control, Fig. 4A) showed no specific staining. To assess the blockade of bADDL binding to neurons, each antibody was then pre-incubated with bADDLs prior to the addition to hippocampal cells. The anti-ADDL antibodies were found to

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block bADDL binding to neurons (Fig. 4E, ACU-966 data not shown), while 6E10 had only a modest effect (Fig. 4F) and A11 and mouse IgG (control, Fig. 4D) did not block binding. Since the biotinylation of ADDLs may have affected the binding of antibodies with an N-terminal epitope such as 6E10, a subsequent study was conducted using ADDLs. The findings were comparable and revealed that the biotinylation of ADDLs did not markedly affect the ability of antibodies to bind neuronally associated ADDLs, although detailed quantitative measures were not employed.

Fig. 4. Primary hippocampal cultures were incubated with a 5 ␮M ADDLs preparation and binding detected with a monoclonal antibody (red). A comparison of the staining pattern seen with ACU-954 (B) and 6E10 (C) revealed a similar pattern of ADDL localization, while no specific staining was seen with a mouse IgG (A, control). The ability of these same antibodies to block the binding of bADDLs to primary hippocampal neurons was then assessed. bADDLs (5 ␮M) were pre-incubated with antibody (5:1 ratio) prior to the addition to primary neuronal cultures and bound bADDLs visualized with a fluorescent labeled streptavidin (red, D–F). A comparison of the efficacy of antibodies to block binding revealed that ACU-954 blocked bADDL binding (E), 6E10 had only a modest effect (F) and mouse IgG (control, D) had no detectable effect on binding. In all figures, the neuronal processes (green) were visualized with a total tau and the nuclei with DAPI (blue). The scale bar is the same for all images.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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3.4. ADDLs bind to neurons in hippocampal slice cultures To explore the relevance of these findings, we investigated the binding of bADDLs to slice cultures from rat hippocampus, a region involved in learning and memory. Initial studies showed that neurons in the CA1-3 (Fig. 5A and B) and dentate gyrus regions were capable of binding ADDLs and bADDLs, while other regions such as the cerebellum did not (data not shown). Moreover, the pattern of binding to processes and the cell soma (Fig. 5B, insert) was similar to that seen in primary cultures (Figs. 2 and 3). When oligomers were preincubated with monoclonal antibodies ACU-954 (Fig. 5C–E) or 6E10 (Fig. 5F–H), the degree of binding was attenuated in a dose-dependent manner. Although both antibodies reduce binding to neurons, ACU-954 blocked binding at a 1:1 ratio (bADDL:antibody) and markedly reduced binding at a 5:1 ratio, while 6E10 was less efficacious. Neither antibody had a detectable effect on binding at a 20:1 ratio, with the binding being comparable to control slices. These results confirmed that bADDLs bind to a subset of neurons in the hippocampus and showed that anti-ADDL and anti-A␤ antibodies can abate this binding, although not to the same extent.

3.5. A quantitative ADDL binding assay While the results of these studies were qualitative and not quantitative in nature, they indicate that antibodies differentially blocked bADDL binding to neurons. To further characterize antibodies, a quantitative alkaline phosphatase assay was developed. Initial studies found a specific, saturable bADDL binding to primary hippocampal neurons (Fig. 6A) and a rat (B103; Schubert and Behl, 1993; Fig. 6B) and mouse neuroblastoma line (N2A). In contrast, no specific binding was seen with an ovarian cell line. Subsequent studies with primary neurons evaluated 11 putative anti-ADDL monoclonal antibodies for their ability to block ADDL binding to neurons. The results revealed that the efficacy of anti-ADDL antibodies varied greatly (Fig. 6C), with some antibodies markedly reducing binding (e.g. ACU-954, ACU-966, and ACU-970), while others were less efficacious (i.e. ACU943). This finding was not predicted based on the shared core linear epitope (aa 3–8 of A␤) of antibodies, thus demonstrating that a conformational epitope is more important for this anti-ADDL activity. Since studies with primary neurons and slice cultures suggested that 6E10 (anti-A␤ antibody) can detect bound ADDLs (Fig. 4C) and, to a lesser degree, reduce

Fig. 5. Assessment of the ability of an anti-A␤ antibody (6E10) and anti-ADDL antibody (ACU-954) to attenuate ADDL binding to hippocampal slice cultures using immunohistochemistry. ADDLs and bADDLs bind to the cell soma and processes (B, insert) of hippocampal CA1 neurons in culture (A and B). When ADDLs or bADDLs (5 ␮M) were pre-incubated with 6E10 or ACU-954 at a ratio of 20:1 (C and F), 5:1 (D and G), or 1:1 (E and H) (ADDLs:antibody) for 1 h, added to slice cultures and the binding visualized (black immunoreactivity), a dose-dependent reduction in binding to the pyramidal cells of Ammon’s horn (CA1 indicated) and the granular cells of the dentate gyrus (DG) was observed. A comparison of antibody blockade indicates that ACU-954 was more efficacious at blocking binding at the 5:1 and 1:1 concentrations, when compared to 6E10. The scale bar is the same for images C–H.

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Fig. 6. Titration curves showing bADDL binding to primary rat hippocampal neurons (A) and rat neuroblastoma cells (B103, B) using an alkaline phosphatase binding assay. Note the linear response of bADDL binding to both cell types. From these bADDLs titration curves, a concentration of 5 ␮M was selected for future antibody blocking experiments (C and D). Subsequent studies evaluated the blockade of bADDL binding to primary hippocampal cells with antiADDL (C and D) or anti-A␤ antibodies (D). A comparison of a panel of anti-ADDL antibodies (C) revealed marked differences in the efficacy of antibodies to block bADDL binding at a ratio of 5:1 (antibody:bADDLs). Some antibodies are highly efficacious in blocking bADDL binding (ACU-954, ACU-966, and ACU-970), others less effective (ACU-914, ACU-942, ACU-947, ACU-959, and ACU-961), and a third group only marginally efficacious (ACU-921, ACU-943, and ACU-988). Subsequently, ACU-954 was compared against a collection of commonly used anti-A␤ antibodies, including antibodies that are known to abate ADDL binding. The results of this comparison (D) demonstrated that the anti-ADDL antibody, ACU-954, was significantly better than any other antibody assessed. Nevertheless, 6E10, 4G8, and G2-11 were found to have a modest effect on bADDL binding to neurons, although not to the same degree as ACU-954. The antibodies A11 and G2-10 (1–40 specific) had no detectable effect on bADDL binding. Error bars represent standard error of the mean.

oligomer binding (Figs. 4F and 5F–H), the anti-ADDL antibody ACU-954 was compared with 6E10 and a collection of other commercial anti-A␤ antibodies (Fig. 6D). The results showed that ACU-954 was significantly better at blocking the binding of bADDLs to neurons than any of the commercial anti-A␤ antibodies assessed (Fig. 6D), although some commercial antibodies (6E10, 4G8, and G2-11) had a modest effect on binding, when compared with ACU-954. In contrast, G2-10 (an A␤1–40 monomer selective antibody), A11 (a putative pan oligomer antibody; Kayed et al., 2003) and mouse IgG (control) were unable to block the binding of bADDLs to neurons under our assay conditions (Fig. 6D). 3.6. ACU-954 blocked ADDL-induced loss of dendritic spines Since bADDLs induce a loss of dendritic spines on hippocampal neurons in culture (Fig. 1D–G) and ACU-954

markedly attenuated bADDL binding to neurons (Figs. 4E, 5C–E, and 6C and D), we assessed the effect of ACU954 at blocking bADDL-induced changes in spine number (Fig. 7). Treatment of the neurons with bADDLs for 24 h induced a marked loss of drebrin-containing spines (Fig. 7A and B). When, however, bADDLs were pre-incubated with ACU-954 for 1 h and then added to neurons (Fig. 7C), a significant reduction in dendritic spine loss (drebrin staining) was noted. In contrast, when denatured ACU-954 was used for studies, no antibody related change in spine loss was detected (compare Fig. 7C and D). Quantification of the drebrin-containing spines from three independent experiments showed a highly significant treatment effect (p < 0.001) among the experimental groups (Fig. 7E). The observation that G2-10, an antibody that was not able to block bADDL binding (Fig. 6D) did not prevent the bADDL-induced loss of spines (data not shown) further demonstrates the specificity of ACU-954 in these studies. These results indicate

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Fig. 7. Changes in the level of drebrin staining on hippocampal dendritic spines after treatment with vehicle (A), bADDLs (B), bADDLs and ACU-954 (C) or bADDLs and denatured ACU-954 (D) were assessed with immunocytochemistry. Note the significant (** p < 0.01) loss of synaptic spines after treatment with bADDLs, when compared with vehicle alone. Pre-incubation of bADDLs with ACU-954, but not denatured ACU-954 attenuated the bADDL-induced loss of drebrin staining (E) and indicated that ACU-954 can abate the morphological changes caused by ADDLs. The scale bar for the low power image and insert are the same for A–D.

that blockade of bADDL binding with ACU-954 functionally protects neurons from synaptic loss caused by A␤ oligomers.

3.7. ACU-954 detects ADDLs in AD cortex Previous studies have shown that anti-ADDL antibodies can stain A␤ complexes in human AD tissue, but not in control brain (Lambert et al., 2007). To assess the ability of ACU-954 to detect naturally occurring oligomers tissue from several confirmed AD patients was evaluated. The results showed specific immunostaining of dense-core plaques (Fig. 8A and B) as well as diffuse deposits (Fig. 8B) throughout the cortical laminae. To confirm the specificity of ACU-954 for ADDLs in the human tissue, the antibody was pre-absorbed with our standard synthetic ADDL preparation. The results of these studies revealed that pre-absorption with a 10× (10:1, ADDL:antibody) preparation (Fig. 8C) dramatically reduced the immunoreactivity, staining that was eliminated when incubated with a 100× ADDLs preparation (Fig. 8D).

4. Discussion The present findings demonstrate that antibodies that detect ADDLs can be identified and characterized, although these antibodies vary greatly in their specificity and selectivity for ADDLs. Importantly, the activity of these anti-ADDL antibodies is not predictable based on the linear epitope on A␤, their ability to detect ADDLs by Western blot or immunostain ADDLs bound to neurons. However, when antibodies were used to immunoneutralize ADDLs and block binding to primary hippocampal neurons, a clear difference was noted; an indication that a more relevant conformational epitope is needed to prevent ADDL binding to neurons. The assessment of a panel of anti-A␤ and anti-ADDL antibodies with this blocking assay identified several anti-ADDL antibodies that were significantly better than others assessed. One antibody, ACU-954 completely blocked the binding of ADDLs to primary neurons and hippocampal slice cultures and was shown to detect naturally occurring oligomers in AD cerebral cortex. Moreover, ACU-954 was able to abate ADDL-induced changes to hippocampal spine morphology, an indication that the blockade of binding has a signifi-

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Fig. 8. The localization of ADDLs in human AD cortex was assessed using an anti-ADDL antibody (ACU-954) and immunocytochemistry. Scattered ACU-954 immunoreactivity was detected throughout the laminae of the cortex (A), with labeling seen in and around dense-core plaques (B), diffuse fibrillar structures (B) and small deposits. The immunoreactivity seen in AD brain with ACU-954 (B) was markedly attenuated when antibody was pre-absorbed with a 10× (C) preparation of synthetic ADDLs (10:1, ADDL:antibody) and eliminated when incubated with a 100× ADDLs preparation (D).

cant physiological ramification. Taken together, the results demonstrate that selective anti-ADDL antibodies can be identified and used to block the binding of ADDLs to hippocampal neurons, a region critically involved in learning and memory. Klein and co-workers were the first to demonstrate that synthetic ADDL preparations (Lambert et al., 1998) as well as ADDLs isolated from Alzheimer’s patients (Gong et al., 2003; Lacor et al., 2004; Lambert et al., 2007) bind to a sub-population of hippocampal and cortical neurons, but not glia, in vitro. Since monomeric A␤ preparations and extracts from normal patients did not bind neurons, they concluded that ADDLs must bind to a cell surface site on specific neuronal populations (Lacor et al., 2004). The results of the present studies have confirmed these earlier observations with ADDLs. In addition, we showed that biotinylated ADDLs (bADDLs) are an exceptional tool to directly assess neu-

ronal binding, as they are visualized with high specificity and affinity and eliminate the reliance on antibodies to detect signal. Interestingly, during the course of these studies, we noted that the number of neurons that bind ADDLs and the degree of neuronal label increased with time in culture. Since the neurons used for primary cultures were procured from embryonic rats (E18) and allowed to “age” for 2–3 wks, the time in culture would encompass the period when neurons are differentiating and forming synapses. In an attempt to better understand ADDL binding to neurons, organotypic hippocampal slice cultures from postnatal day 12 rats were used for binding studies. The results showed specific ADDL binding to the CA1-3 pyramidal neurons in Ammon’s horn and granular cells of the dentate gyrus. Subsequent studies revealed that both GABAergic and glutamatergic hippocampal neurons bind ADDLs. These observations, and the finding

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that ADDLs bind to neuroblastoma (B103 and N2A), but not an ovarian line (CHO), demonstrate that ADDL binding is not ubiquitous to all cells and tissues. Furthermore, these findings argue that ADDLs are not randomly intercalating into membranes to form pores in a non-specific manner (see Lin et al., 2001; Lashuel et al., 2002). Rather, ADDLs appear to bind to a cell surface protein(s) that is/are developmentally expressed in a sub-population of neurons (e.g. hippocampus), with the onset of expression correlating with neuronal maturation and synapse formation. While several members of the NMDA receptor family have been suggested as putative binding partners for ADDLs, a definitive “receptor” remains to be described (De Felice et al., 2007). Previous studies (Lambert et al., 1998; Dahlgren et al., 2002; Klyubin et al., 2005; Ma et al., 2006; Lesne et al., 2006) showed that the anti-A␤ antibodies 6E10 and 4G8 detect naturally occurring and synthetic ADDLs by Western or dot blot. In addition, 6E10 can abate ADDL-induced changes in spine density (Shankar et al., 2007) and can attenuate ADDL-induced deficits in LTP when co-administered with ADDLs into the rodent brain (Klyubin et al., 2005). Initial studies, described herein, revealed that pre-incubation of ADDLs or bADDLs with 6E10 had a modest effect on binding to neuroblastoma cells, primary hippocampal neurons, and hippocampal slice cultures. In an attempt to better understand these antibodies and extend our observations, we compared 6E10 with an anti-ADDL antibody (ACU954) in several cell-based assays. Both ACU-954 and 6E10 detected ADDLs bound to a sub-population of neurons, with a comparable degree of immunostaining. However, when the antibodies were pre-incubated with bADDLs prior to binding assessment with immunofluorescence, a clear distinction was observed. ACU-954 completely blocked the binding of bADDLs to neurons, whereas 6E10 had only a modest effect on this association. Additional studies with ADDLs confirmed that the N-terminal biotin-label on bADDLs was not responsible for the differential effect of ACU-954 and 6E10 on binding. The results of these studies revealed that 6E10 had some activity in blocking ADDL binding to neurons, but not to the same degree as several anti-ADDL antibodies. Although the results of these binding studies and previous observations (Gong et al., 2003; Lambert et al., 2007) were qualitative in nature, they clearly indicated that antibodies differentially block ADDL binding to neurons. To further assess and characterize a larger collection of putative anti-ADDL monoclonal antibodies, a quantitative 96-well alkaline phosphatase assay was developed. The comparison of 11 anti-ADDL antibodies showed that each antibody was unique in its ability to block ADDL binding to primary hippocampal neurons, with some antibodies (i.e. ACU-954, ACU-966, and ACU-970) being significantly more efficacious than others (i.e. ACU-921, ACU-943, and ACU-988) despite the fact that the antibodies had the same predicted core linear epitope. A subsequent study compared ACU-954 with a panel of commonly used commercial antibodies (anti-A␤ and a pan anti-oligomer). The results revealed three distinct groups of

antibodies; ones that markedly attenuated binding (ACU954), those that had a modest effect on binding (6E10, 4G8, and G2-11) and antibodies that were not different from control (G2-10 and A11). The results from this study confirmed our previous immunocytochemistry findings that showed a modest effect of 6E10 on reducing ADDL binding. Since 6E10, 4G8, and G2-11 appear to detect all forms of A␤ (i.e. monomeric, ADDLs, and fibrillar), it is likely that the efficacy of these antibodies to block ADDL binding is related to their specificity and avidity for ADDLs. The finding that G2-10, an A␤1–40 monomer selective antibody was unable to block binding was expected, since we had shown previously that only high molecular weight species (oligomers) bind neurons (Hepler et al., 2006). However, the finding that A11 was unable to detect bound ADDLs or block the binding of ADDLs to neurons was unexpected, since A11 has previously been shown to detect synthetic oligomers (Kayed et al., 2003) as well as oligomers in a 3xTg-AD mouse (Oddo et al., 2006) and in human AD brain (Kokubo et al., 2005). A careful comparison of our ADDL preparations with oligomers generated using other methods indicated that the size and stability of high molecular weight species varied among protocols. This observation suggests that A11 may recognize an oligomeric species that is enriched in some preparations, but sparse in our ADDLs and bADDLs. Despite the lack of A11 immunoreactivity, the present findings demonstrate that ADDLs and bADDLs bind hippocampal neurons and induce morphological changes to dendritic spines, changes that are thought to effect synaptic function. A growing body of evidence suggests that oligomers of A␤ (ADDLs), and not plaques, play a fundamental role in the cognitive decline that is typically associated with Alzheimer’s disease (see Walsh and Selkoe, 2004). ADDLs are elevated in AD brain and induce deficits in behavioral and electrophysiological endpoints when centrally administered to rodents (Walsh et al., 2002; Cleary et al., 2005; Klyubin et al., 2005). Deficits in learning and memory have also been observed in a hAPP expressing mouse model, with the onset of impairment associated with elevated ADDL levels (Westerman et al., 2002; Ashe, 2005; Lee et al., 2005; Lesne et al., 2006). While the cellular and sub-cellular events that mediate these effects on cognition are not fully understood, it is clear that ADDLs bind to the synaptic terminals localized on the dendritic processes of hippocampal neurons (Lacor et al., 2004) and alter the morphology and number of dendritic spines (Lacor et al., 2007; Shankar et al., 2007). The present findings that ADDLs bind to both GABAergic and glutamate neurons in the hippocampus, neurons critically involved in learning and memory, further bolster the argument that ADDLs could directly or indirectly modulate these neurotransmitter systems (see Venkitaramani et al., 2007). Additionally, the observation that ACU-954 stains plaques in the AD brain, a pattern that is blocked when antibody is pre-incubated with ADDLs, suggests that ADDLs may associate with plaques in the AD brain as well. This possibility is supported by the recent findings that exogenous bADDLs accumulate in

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cortical and hippocampal dense-core plaques when administered to mice that over express hAPP (Gaspar, Bowles and Shughrue, unpublished observations). Future studies are clearly needed to ascertain the binding site(s) and repositories for ADDLs and determine the immediate impact of ADDL binding on neuronal function, even though several candidates have been proposed. ADDL binding may also have long-term effects on neurons. Recent studies have shown that ADDL binding to hippocampal neurons can initiate a signaling cascade that results in the phosphorylation of tau (De Felice et al., 2007). One component of this signaling cascade, GSK-3␤ has also been shown to be modulated by ADDL binding in vivo and in vitro (see Ma et al., 2006). Interestingly, Ma et al. (2006) found that the passive immunization of hAPP mice with an antibody that reduced ADDLs, also reduced GSK-3␤ levels and the phosphorylation of tau in the cortex. These findings support a link between A␤ and phosphorylated tau and suggest that ADDL binding may trigger events that lead to the intracellular aggregation of tau. Furthermore, the data suggests that the identification of antibodies that prevent the binding of ADDLs to neurons and the associated loss of synaptic spines (such as ACU-954) could alleviate some of the cognitive and/or pathological outcomes associated with Alzheimer’s and related diseases. Disclosure statement All authors were employed by Merck and Company at the time of these studies. Acknowledgements We gratefully acknowledge Grant Krafft, PhD (Acumen Pharmaceuticals) for providing the panel of anti-ADDL antibodies and the M71 used in these studies, Sethu Sankaranarayanan, PhD, Eric Price, MS and Adam Simon, PhD (Alzheimer’s Research, Merck) for their early contributions to setting up the primary cell cultures and ADDL binding methods, Debbie Nahas, BS (Vaccine and Biologics Research, Merck) for the generation of ADDL and bADDL preparations and Renee Gaspar, MS (Alzheimer’s Research, Merck) for her technical assistance with confocal microscopy. We are also in debt to Paul Keller, PhD (Vaccine and Biologics Research, Merck), Xiaoping Liang, PhD (Vaccine and Biologics Research, Merck) and Guy Seabrook, PhD (Alzheimer’s Research, Merck) for their invaluable advice throughout these studies and thoughtful criticism during the preparation of this manuscript. References Ashe, K.H., 2005. Mechanisms of memory loss in A␤ and tau mouse models. Biochem. Soc. Trans. 33, 591–594.

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