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Originally published In Press as doi:10.1074/jbc.M511018200 on December 16, 2005

J. Biol. Chem., Vol. 281, Issue 7, 4292-4299, February 17, 2006
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Targeting Amyloid-beta Peptide (Abeta) Oligomers by Passive Immunization with a Conformation-selective Monoclonal Antibody Improves Learning and Memory in Abeta Precursor Protein (APP) Transgenic Mice*Formula

Edward B. Lee{ddagger}, Lewis Z. Leng{ddagger}, Bin Zhang{ddagger}, Linda Kwong{ddagger}, John Q. Trojanowski{ddagger}§1, Ted Abel, and Virginia M.-Y. Lee, The John H. Ware III professor of Alzheimer research{ddagger}§2

From the {ddagger}Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, the §Institute on Aging, University of Pennsylvania School of Medicine, and the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, October 11, 2005 , and in revised form, December 5, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Passive immunization of murine models of Alzheimer disease amyloidosis reduces amyloid-beta peptide (Abeta) levels and improves cognitive function. To specifically address the role of Abeta oligomers in learning and memory, we generated a novel monoclonal antibody, NAB61, that preferentially recognizes a conformational epitope present in dimeric, small oligomeric, and higher order Abeta structures but not full-length amyloid-beta precursor protein or C-terminal amyloid-beta precursor protein fragments. NAB61 also recognized a subset of brain Abeta deposits, preferentially mature senile plaques, and amyloid angiopathy. Using NAB61 as immunotherapy, we showed that aged Tg2576 transgenic mice treated with NAB61 displayed significant improvements in spatial learning and memory relative to control mice. These data implicated Abeta oligomers as a pathologic substrate for cognitive decline in Alzheimer disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Abeta peptide has been hypothesized to cause the pathologic and behavioral manifestations of Alzheimer disease (AD),3 including synaptic dysfunction and loss, neurofibrillary tangle formation, neuronal degeneration, and impaired memory. A variety of methods designed to inhibit the production or enhance the clearance of Abeta are being developed as potential AD therapies. Indeed, immunization of murine models of Abeta amyloidosis inhibits senile plaque formation and ameliorates associated cognitive impairments (1–6). Despite the development of meningoencephalitis in 6% of individuals immunized with the Abeta42 peptide during a phase II clinical human trial (7, 8), immunotherapy, especially passive immunization, remains a compelling potential treatment for AD. Interestingly, passive immunization of mouse models of AD-like Abeta plaques has been shown to rapidly reverse learning and memory deficits without affecting Abeta plaque pathology, indicating that neutralization of toxic Abeta species can quickly restore neuronal function in vivo (9, 10).

The lack of learning and memory deficits in young APP transgenic mice indicates that monomeric Abeta is not responsible for behavioral impairments in vivo (11, 12). Furthermore, levels of soluble monomeric Abeta do not increase with age or with the onset of cognitive defects in transgenic mice (13). Therefore, if Abeta is responsible for learning and memory deficits in vivo, then Abeta must gain one or more of its toxic properties as a function of time. One potential mechanism for this toxic gain of function is a change in the conformation of Abeta such that it exerts its pathologic effects as an oligomeric or fibrillar macromolecule.

To specifically target toxic forms of Abeta, we developed a monoclonal antibody named NAB61 that recognizes a pathologic conformation present in Abeta dimers, soluble oligomers, and higher order species of Abeta. Using this antibody, we found that neutralization of pathologic Abeta by passive immunization of transgenic mice resulted in rapid improvement in spatial learning and memory. These results suggest that pathologic Abeta conformers produced in vivo are capable of disrupting neuronal function, and our data have substantiated the therapeutic potential of targeting Abeta oligomers for the treatment of AD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of NAB61—Synthetic Abeta1–40 (from D. Teplow, Boston, MA or from W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT) was treated with peroxynitrite, UV light, or 4-hydroxynonenal (HNE) as described (14–16). BALB/c mice were immunized with 100 µg of these Abeta species emulsified with complete Freund's adjuvant followed by three additional injections of 25 µg of Abeta emulsified with incomplete Freund's adjuvant. Isolated lymphocytes were used to generate hybridomas by fusion with Sp2/O-Ag14 myeloma cells with polyethylene glycol 1500.

Immunoprecipitation and Immunoblotting—Synthetic Abeta preparations (0.5 µg) were electrophoresed on 16% Tris-Tricine gels and immunoblotted with NAB228 or NAB61 or immunoprecipitated using NAB61 or NAB228 with protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) prior to immunoblotting with NAB228. For radiolabeled APP and C-terminal APP fragments, CHO Pro5 cells transfected with pcDNA3.1 containing the cDNA for either green fluorescent protein or APP harboring the Swedish Mutation (APPswe) were radiolabeled with [35S]methionine for 2 h in the presence of 200 µM MG132 (Peptides International, Louisville, KY) to enhance the accumulation of C-terminal APP fragments. RIPA buffer cell lysates (0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 5 mM EDTA in Tris-buffered saline, pH 8.0) containing protease inhibitors (1 µg/ml pepstatin A, leupeptin, L-1-tosylamido-2-phenylethyl chloromethyl ketone, 1-chloro-3-tosylamido-7-amino-2-heptanone, and soybean trypsin inhibitor and 0.5 mM phenylmethylsulfonyl fluoride) were centrifuged at 100,000 x g for 20 min at 4 °C and immunoprecipitated with a panel of monoclonal antibodies that recognize Abeta1–11 (NAB14, NAB61, NAB89, NAB228, Ban50), a rabbit polyclonal antibody raised against the C terminus of APP (antibody number 2493), or a goat polyclonal antibody raised against the N-terminal ectodomain of APP (Karen). NAB antibodies were generated using the oligomeric Abeta preparations as antigens but do not exhibit oligomer specificity. Immunoprecipitates were electrophoresed on 10/16.5% step gradient Tris-Tricine gels, fixed with methanol, dried, and exposed to a phosphorimaging screen for visualization. APP and APP fragments from mouse cortical extracts were detected as described (17), using the following antibodies: rabbit polyclonal antibody raised against the C terminus of APP (antibody number 5685); goat anti-N-terminal APP antibody (Karen); rabbit anti-sAPPbetaswe (antibody number 54); and NAB228. An anti-tubulin antibody (TUB2.1, Sigma) was used to detect tubulin. To detect peripheral Abeta, 200 µl of plasma was diluted with RIPA buffer and immunoprecipitated with 4G8 (anti-Abeta17–24), which was covalently conjugated to protein A/G beads with dimethyl pimelimidate to prevent competition with endogenous IgG. Immunoprecipitates were electrophoresed on a 10/16.5% Tris-Tricine gel and immunoblotted with 4G8.

Immunocytochemistry and Immunoelectron Microscopy—Neuro2A, CHO Pro5, and NT2N neurons were transduced with a Simliki Forest Virus encoding APPswe (SFV-APPswe). After 16 h, cells were fixed with cold 95% ethanol, 5% acetic acid for 10 min followed by further permeabilization with 0.2% Triton X-100 in PBS for 10 min. Cells were stained with a goat polyclonal N-terminal APP antibody (Karen) and NAB61 followed by fluorescein isothiocyanate-conjugated anti-goat IgG and Texas Red-conjugated anti-mouse IgG. For immunoelectron microscopy, fibrillar synthetic Abeta40 was absorbed to 300 mesh carbon-coated copper grids, washed with PBS, and blocked with 1% bovine serum albumin in PBS. Abeta fibrils were stained with NAB61 followed by anti-mouse IgG conjugated to 5-nm colloidal gold particles. Grids were then stained with 1% uranyl acetate, dried, and visualized with a Joel (Peabody, MA) 1010 transmission electron microscope.

Immunohistochemistry—Tissue blocks from human subjects or mice were immersion-fixed in 70% ethanol with 150 mM NaCl or 10% buffered formalin. Samples were dehydrated through graded ethanol solutions to xylene and infiltrated with paraffin as described (18). Sections (6 µm) were stained using standard avidin-biotin-peroxidase methods using 3-3'diaminobenzidine. Ban50 (mouse anti-Abeta1–10), NAB228 (mouse anti-Abeta1–11), and NAB61 (mouse anti-oligomer Abeta1–11) were used as primary antibodies followed by HRP-conjugated anti-mouse secondary antibody (Vector, Burlingame, MD). Quantification of Abeta plaque burden in the Tg2576 mice overexpressing human APP harboring the Swedish mutation (11) was conducted as described (17). For immunofluorescence, sections were stained with a rabbit polyclonal anti-Abeta42 antibody (BIOSOURCE International, Camarillo, CA) and with NAB61 followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG and Texas Red-conjugated anti-mouse IgG.

ELISA Analysis—For solid-phase experiments, ELISA plates (Nunc, Rochester, NY) were coated with Abeta at 1 µg/ml in PBS and blocked with 5% fetal bovine serum in PBS. Antibodies diluted in 5% fetal bovine serum/PBS were incubated at 4 °C overnight, and bound antibodies were detected with HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). For blocking experiments, ELISA plates were coated with Abeta at 0.1 µg/ml, and antibodies were preincubated with the indicated blocking peptides at 10 µg/ml. For capturing experiments, ELISA plates were coated either NAB61 or Ban50 at 10 µg/ml in PBS and blocked with 1% casein in PBS. Abeta peptides diluted in blocking buffer at 10 µg/ml were incubated at 4 °C overnight, and bound Abeta was detected with HRP-conjugated BA27 (mouse anti-Abeta40).

For Abeta quantification, detergent-soluble fractions of cortical and hippocampal regions were obtained by sonicating samples in 1 ml of RIPA buffer containing protease inhibitors for every 150 mg of tissue. After centrifugation at 100,000 x g for 20 min at 4 °C, the resulting pellet was solubilized by sonication in 70% formic acid followed by another round of centrifugation to obtain detergent-insoluble Abeta. Supernatants were assayed by sandwich ELISA as described previously (19). Briefly, ELISA plates were coated with either JRF/c40 or JRF/c42 to capture Abeta40 and Abeta42, respectively. After application of diluted samples and a standard curve consisting of serially diluted synthetic Abeta (Bachem Biosciences, King of Prussia, PA), the concentration of Abeta was determined by using horseradish peroxidase-conjugated m266 (anti-Abeta13–28) as a reporting antibody. These antibodies do not recognize the N terminus of Abeta and therefore do not compete with NAB61 for Abeta.

Immunization and Behavioral Analysis—Tg2576 mice were maintained on a C57B6/SJL F2 background by successive backcrossing to wild-type C57B6/SJL F1 females. All mice were generated and handled according to University of Pennsylvania Institutional Animal Care and Use Committee guidelines. To determine whether immunization improves learning and memory, 17–19-month-old Tg littermates were administered an initial dose of 400 µg of NAB61 or nonspecific mouse IgG (Sigma) intraperitoneally followed by maintenance doses of 200 µg as described in Fig. 5A. Wild-type littermates were administered either IgG or NAB61, and these two wild-type groups were analyzed as one group after treatment was determined to have no effect in non-Tg mice as described under "Results." The visible and hidden versions of the Morris water maze (MWM) were performed as described by Westerman et al. (12) and are described in detail in the Supplemental Methods. Briefly, mice were subjected to the visible platform MWM in which latency to reach a visibly marked platform was measured over six consecutive training blocks (four trials/block, two blocks/day). Mice were then subject to the hidden platform MWM in which the latency to reach a submerged platform was measured over 9 days (four trials/block, one block/day). Three probe trials were interspersed throughout the hidden platform MWM to test for acquisition of visual reference memory by removing the hidden platform and recording swimming behavior for 60 s. Data were recorded using a video tracking system (HVS Image, San Diego, CA). Mice were tested in a blinded manner, with groups balanced for genotype and treatment. Mice were sacrificed 3 days after the termination of the MWM for biochemical and histological assessment. Experiments were analyzed with one-way ANOVA and two-way ANOVA, with Fischer's test for post-hoc analysis. To determine whether passive immunization removed Abeta plaques, long term passive immunization was performed on a cohort of 8-month-old Tg2576 mice, which were administered weekly doses of intraperitoneal NAB61 or nonspecific IgG (500 µg) for 6 months and sacrificed at 14 months of age. Changes in Abeta deposits by immunohistochemistry and Abeta levels by ELISA were conducted as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of an Oligomer-selective Monoclonal Antibody—The role of Abeta oligomers is difficult to assess in vivo due to the dearth of conformation-specific molecular tools. Toward this end, we generated a monoclonal antibody that selectively recognizes oligomeric Abeta by using a stable oligomeric Abeta preparation as antigen. Treating synthetic Abeta1–40 with peroxynitrite, a reactive species generated by the reaction between superoxide and nitric oxide, resulted in the formation of SDS-stable Abeta oligomers, as shown by SDS-PAGE followed by immunoblotting with NAB228, a monoclonal antibody that recognizes a linear N-terminal Abeta epitope (Fig. 1A). Additional stable Abeta oligomers were also generated by treating Abeta1–40 with UV light and with the lipid-derived reactive aldehyde, 4-HNE (gifts from D. Teplow and T. Montine). Over 5,500 hybridoma supernatants were tested for the presence of Abeta antibodies, and one hybridoma was generated from a mouse immunized with nitrated Abeta that produced an IgG1, named NAB61, with selectivity toward oligomeric Abeta species. As shown by both immunoblotting and immunoprecipitation, NAB61 showed selectivity toward SDS-stable Abeta oligomers relative to monomeric Abeta (Fig. 1, A and B, left panels), in contrast with other Abeta antibodies such as NAB228 (Fig. 1, A and B, right panels).


Figure 1
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  		FIGURE 1.
Conformational epitope of NAB61, an Abeta oligomer-selective monoclonal antibody. A, NAB61 preferentially recognizes Abeta oligomers by immunoblotting. Abeta1–40 was treated with a peroxynitrite, UV light, or 4-HNE, electrophoresed on a 16% Tris-Tricine gel, and blotted with either NAB61 (left) or NAB228 (right). B, NAB61 preferentially recognizes Abeta oligomers by immunoprecipitation. Abeta1–40 was treated with a peroxynitrite, UV light, or 4-HNE and subject to immunoprecipitation with NAB61 (left) or NAB228 (right). Immunoprecipitates were electrophoresed on a 16% Tris-Tricine gel and blotted with NAB228. C, solid-phase immunoreactivity of NAB61. NAB61, NAB228, and nonspecific mouse IgG were serially diluted and tested for immunoreactivity on ELISA plates coated with Abeta1–40 (1 µg/ml). D, NAB61 recognizes an N-terminal epitope. Solid-phase ELISA immunoreactivity was blocked by preincubation of NAB61 and NAB228 with peptides corresponding to full-length Abeta (residues 1–40), the N terminus of Abeta (residues 1–11), or the C terminus of Abeta (11–40). Only peptides containing the N terminus of Abeta were able to block NAB61 and NAB228 immunoreactivity. E, NAB61 preferentially recognizes oligomeric Abeta when used in a sandwich ELISA as a capturing antibody. ELISA plates coated with either Ban50 or NAB61 were tested for their ability to capture Abeta or UV-cross-linked Abeta. Captured peptides were detected with HRP-conjugated BA27 (anti-Abeta40). A two-tailed t test was performed on OD readings performed in duplicate from two independent experiments (***, p < 0.001).

 
To further characterize this novel monoclonal antibody, we tested NAB61 in a variety of additional immunologic assays. When tested in a solid-phase ELISA format in which Abeta1–40 was coated onto plastic, NAB61 titers were very low relative to NAB228 (Fig. 1C). Despite this low immunoreactivity, peptides corresponding to Abeta1–11 and Abeta1–40 were able to block the signal generated by NAB61, whereas Abeta11–40 did not, indicating that NAB61 recognizes an N-terminal Abeta epitope (Fig. 1D). When used as a capturing antibody in a sandwich ELISA format, NAB61 had greater affinity for oligomeric Abeta relative to non-oligomeric Abeta, in contrast with other anti-Abeta monoclonal antibodies such as Ban50 (Fig. 1E). These in vitro studies indicated that NAB61 recognizes a complex conformational epitope found in the N terminus of oligomeric forms of Abeta.

Although the primary sequence of Abeta is present in full-length APP and C-terminal APP fragments, an antibody that recognizes a pathologic Abeta conformation should be specific for the Abeta peptide. Therefore, we hypothesized that NAB61 does not recognize full-length APP or C99, akin to human anti-Abeta antibodies generated upon active immunization (20). Immunoprecipitations from radiolabeled CHO cells overexpressing either green fluorescent protein or APP were performed with a panel of monoclonal antibodies that recognize the N terminus of Abeta (designated NAB antibodies), a polyclonal N-terminal APP antibody, and a polyclonal C-terminal APP antibody. All of the NAB antibodies recognized both full-length APP and C99 with the notable exception of NAB61 (Fig. 2A). The lack of cross-reactivity with APP was confirmed by double immunofluorescence staining of Neuro2a, NT2N, and CHO cells overexpressing APPswe, which showed that NAB61 staining did not co-localize with staining of total APP by a polyclonal N-terminal APP antibody (Fig. 2B). Therefore, NAB61 recognizes a conformational epitope specific to oligomeric Abeta, which is not present in the Abeta sequence when found in the context of APP or C99.


Figure 2
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  		FIGURE 2.
NAB61 does not recognize full-length APP or C99. A, CHO Pro5 cells were transfected with either green fluorescent protein (GFP)(left) or APPswe (right) and radiolabeled with [35S]methionine for 2 h. RIPA lysates were immunoprecipitated with a panel of N-terminal monoclonal antibodies (NAB14, NAB61, NAB89, NAB228, and Ban50), a polyclonal antisera raised against the C terminus of APP, and a polyclonal antisera raised against the N-terminal ectodomain of APP. Immunoprecipitated material was electrophoresed on a 10/16.5% Tris-Tricine gel and visualized by exposure to a phosphorimaging screen. B, Neuro2A, NT2N neurons, and CHO Pro5 cells were transduced with an SFV-APPswe vector and subject to double immunofluorescence using an N-terminal APP polyclonal antibody (Karen; green) and NAB61 (red). A merged image is shown on the right with a 4',6-diamidino-2-phenylindole counterstain for nuclei (blue).

 
NAB61 Immunoreactivity against Fibrillar Abeta Amyloid—To ensure that NAB61 recognized bona fide Abeta amyloid, immunohistochemistry was performed on a variety of tissues containing Abeta amyloid plaques. Individuals with pathologic aging (no history of cognitive impairment despite the presence of Abeta amyloid deposits), mild cognitive impairment (MCI) as demonstrated by psychometric testing, Down syndrome, and AD all contained Abeta plaques and other Abeta deposits that were NAB61-immunoreactive (Fig. 3). Interestingly, diffuse amyloid plaques were poorly stained by NAB61, despite robust staining of amyloid angiopathy (see Fig. 3, insets, for pathologic aging). Compact amyloid plaques from Tg2576 transgenic mice overexpressing APPswe were also recognized by NAB61 (Fig. 3).

The initial stages of AD pathology are generally characterized by Abeta amyloid pathology in association cortices such as the mid-frontal cortex. With disease progression, Abeta amyloid in neocortical regions becomes more advanced, co-incident with the development of relatively milder Abeta deposits in limbic regions such as the entorhinal cortex and the hippocampus (21). NAB61 immunoreactivity generally exhibited regional selectivity, which reflected the regional progression and severity of Abeta amyloid pathology. For example, many diffuse amyloid deposits in the hippocampus and entorhinal cortex of AD brains were poorly stained by NAB61 (Fig. 4A, left and middle panels) despite strong staining of mature senile plaques and amyloid angiopathy (Fig. 4A, arrowheads, and 4B), whereas many Abeta amyloid plaques in the mid-frontal cortex showed more robust NAB61 immunoreactivity (Fig. 4A, right panels).


Figure 3
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  		FIGURE 3.
NAB61 recognizes Abeta amyloid deposits. Sections from a variety of pathologic specimens were stained with Ban50/NAB228 (left) or NAB61 (right) and counterstained with hematoxylin for nuclei. Individuals categorized as pathologic aging have no clinical history of cognitive impairment but do have abundant cerebral Abeta deposits. Insets show a higher magnification view of a blood vessel with amyloid angiopathy (left) or a diffuse Abeta deposit (right) from the mid-frontal cortex. Also shown are sections from the mid-frontal cortex of individuals with MCI, Down syndrome, and AD. Dense deposits found in brains of Tg2576 mice were also recognized by NAB61. All images were taken from serial sections with the exception of the individual with MCI.

 
The regional selectivity of NAB61 immunoreactivity suggested that NAB61 recognizes a conformation that is found in advanced, pathologic Abeta deposits, namely mature senile plaques and amyloid angiopathy. These inclusions are distinct from diffuse Abeta deposits, which are not associated with neuritic alterations, tau pathologies, or neuronal loss. Double immunofluorescence staining showed that many amorphous Abeta deposits, which were recognized by a conventional anti-Abeta42 antibody, were not recognized by NAB61 (Fig. 4B). In contrast, mature senile plaques were labeled by both antibodies (Fig. 4B). Given that Abeta fibrils are the ultrastructural building blocks of senile plaques, NAB61 staining of synthetic Abeta fibrils by immunoelectron microscopy further corroborated the ability of NAB61 to recognize pathologic forms of Abeta (Fig. 4B). Therefore, NAB61 appears to recognize a pathologic conformation present in dimeric and oligomeric Abeta, which is maintained during fibrillization and coalescence into senile plaques. NAB61 did not recognize other inclusions consisting of amyloidogenic proteins such as neurofibrillary tangles or Lewy bodies (data not shown), indicating that NAB61 is specific for Abeta and does not recognize a pathologic conformation common to other amyloidogenic proteins.


Figure 4
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  		FIGURE 4.
NAB61 recognizes a subset of fibrillar Abeta pathology. A, regional distribution of NAB61 immunoreactivity. Sections from the hippocampus, entorhinal cortex, and mid-frontal cortex from individuals with AD display different amounts of NAB61 immunoreactivity. In cases of AD in which limbic regions contain predominantly diffuse-type Abeta deposits, NAB61 immunoreactivity was not abundant, although amyloid angiopathy and dense senile plaques were strongly stained (arrowheads). In contrast, abundant staining was observed in regions such as the mid-frontal cortex, where dense, mature senile plaques predominate. B, a higher magnification view of serial sections from the entorhinal cortex of an individual with AD clearly demonstrates that NAB61 does not recognize all Abeta deposits (left panels). Double immunofluorescence with a polyclonal anti-Abeta42 antibody (green) and NAB61 (red) of the entorhinal cortex demonstrates that NAB61 recognizes a mature, dense, cored senile plaque. In contrast, a diffuse Abeta deposit was not stained by NAB61 (upper right panels). NAB61 recognizes Abeta fibrils by immunoelectron microscopy. NAB61 staining of synthetic Abeta1–40 fibrils was detected with 5 nm colloidal gold-conjugated anti-mouse IgG (lower right panel).

 
NAB61 Improves Spatial Learning and Memory—Learning and memory impairments have been shown to normalize rapidly upon neutralization of Abeta following passive immunization of murine models of Abeta amyloidosis (9, 10). To probe the role of Abeta oligomers on cognitive dysfunction, 17–19-month-old Tg2576 mice were immunized with NAB61 (n = 14) or nonspecific IgG (n = 16) and tested in the MWM for spatial learning and memory, using the schedule shown in Fig. 5A. Non-transgenic mice were also treated with either IgG (n = 7) or NAB61 (n = 7). However, for statistical analysis, data from IgG- and NAB61-treated non-transgenic control mice were pooled after performing a two-way ANOVA, which revealed no effect of treatment on performance in the hidden water maze for non-Tg mice (treatment, p = 0.8974; block, p = 0.0251; interaction, p = 0.8942).

To test for potentially confounding sensorimotor or motivational defects, latencies to reach a visible platform were measured over successive training blocks (four trials/block). Non-transgenic, NAB61-treated Tg2576, and IgG-treated Tg2576 mice showed no deficits in the visible water maze (Fig. 5B). Although latencies for both NAB61-treated and IgG-treated Tg2576 mice on the second and third training blocks of the visible water maze tended to be higher than latencies for non-transgenic mice, differences in overall performance were insignificant (repeated measures ANOVA p = 0.0814). Furthermore, the latency to reach the visible platform and swim speeds on both the first trial and the first block were not statistically different between the three groups (data not shown), arguing against the presence of confounding sensorimotor or motivational deficits.

Spatial learning and memory were then tested using the hidden water maze in which the primary measure of learning and memory was latency to reach the hidden platform (Fig. 5C). IgG-treated Tg2576 mice showed no significant improvement in latencies over the testing period (one-way ANOVA, p = 0.4402), whereas NAB61-treated Tg2576 mice and non-transgenic mice both showed a significant decrease in latency with training (one-way ANOVA: NAB61, p = 0.0004; non-transgenic, p = 0.0184). Furthermore, comparisons between the three groups of mice demonstrated that both non-transgenic and NAB61-treated Tg2576 mice performed significantly better than IgG-treated Tg2576 mice (repeated measures ANOVA, p = 0.0002; non-transgenic versus IgG, p = 0.0014; NAB61 versus IgG p = 0.0006). Therefore, passive immunization with NAB61 ameliorates behavioral deficits in the hidden water maze.

To confirm that the improved behavior on the hidden water maze was due to the acquisition of spatial reference memory, three probe trials were interpolated throughout the training process (Fig. 5A) in which the platform was removed, and the percentage of time spent searching in the target quadrant (where the platform is usually located) was determined. During the first probe trial, the three groups of mice exhibited spatially oriented swimming behavior, indicating that all three groups have acquired some degree of a spatial reference for the general location of the hidden platform (Fig. 5D). However, the time spent in the target quadrant relative to adjacent quadrants was only significantly different for NAB61-treated Tg2576 and non-transgenic mice. After further training, this behavioral measure became saturated and thus was unable to discern any differences between the three groups of mice in the final two probe trails (Fig. 5, E and F).

Since the time spent in the target quadrant appeared to plateau by the second of the three probe trials, we used a third measure of spatial learning to confirm the improved acquisition of spatial reference memory upon NAB61 immunization. A platform crossing index was calculated that measures the number of crossings over the exact location of the platform subtracted by the average number of crossings over the platform locations in non-target quadrants (Fig. 5G). Using this measure, both NAB61-treated Tg2576 mice and non-transgenic mice performed significantly better than IgG-treated Tg2576 mice (repeated measures ANOVA, p = 0.0301; NAB61 versus IgG, p = 0.0332; non-transgenic versus IgG, p = 0.0426). Again, this effect was not due to the presence of motor deficits as all three groups of mice exhibited similar swim speeds regardless of the probe trial (Fig. 5H, two-way ANOVA: group, p = 0.4033; probe trial, p = 0.8911; interaction, p = 0.9804). Therefore, three statistical measures (latency, percentage of time in target quadrant, and platform crossing index) all indicated that short term immunization with NAB61 improved spatial learning and memory in aged Tg2576 mice.

NAB61 Immunization Does Not Affect APP Processing or Abeta Accumulation—To show that the NAB61-mediated neutralization of Abeta oligomers was independent of effects on APP processing or the extent of Abeta amyloid pathology, we examined levels of APP and APP fragments in mice after NAB61 treatment. Steady-state levels of full-length APP, sAPPbeta, and C99 were not different among IgG-treated and NAB61-treated Tg2576 mice, arguing that NAB61 did not affect proteolytic processing of APP (Fig. 6A). Furthermore, no differences in amyloid plaque morphology, distribution, or density were noted upon NAB228 or NAB61 immunohistochemistry (Fig. 6B). Additionally, quantification of detergent-soluble and insoluble levels of cortical and hippocampal Abeta by sandwich ELISA indicated that Abeta levels were not statistically different upon short term NAB61 immunization (Fig. 6C). Surprisingly, we also found that 6 months of peripheral passive immunization (Table 1) or direct intracerebral inoculation (supplemental Table 1) performed in additional cohorts of Tg2576 mice did not reduce Abeta amyloid burden, further arguing that the effect of NAB61 on cognitive behavior was via direct neutralization of Abeta oligomers and not on APP processing or Abeta amyloid pathology.


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  		TABLE 1
Abeta quantification of Tg2576 mice immunized with NAB61 Tg2576 mice were treated with weekly intraperitoneal injections of 500 µg of non-specific IgG (n = 8) or NAB61 (n = 9) from 8 to 14 months. Detergent-soluble and insoluble fractions were obtained from the cortex and hippocampus (Hipp) of treated mice by sequential extraction with RIPA buffer and formic acid.

 


Figure 5
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  		FIGURE 5.
NAB61 improves spatial learning and memory. A, immunization and behavioral testing. 17–19-month-old Tg2576 mice were administered an initial dose of 400 µg of NAB61 or nonspecific mouse IgG on day -3 with maintenance doses of 200 µg on days 0, 6, and 12. Performance on the visible water maze was tested on days 1–3 (four trials/block, two blocks/day) followed by the hidden water maze on days 4–13 (four trials/block, one block/day). Probe trials were interpolated throughout the hidden water maze on days 7, 10, and 13. B, performance in the visible water maze. NAB61-treated Tg2576, IgG-treated Tg2576, and non-transgenic mice all learned the visible water maze task. No significant differences were found between the three groups. C, performance in the hidden water maze. Within-group analysis indicated that NAB61-treated Tg2576 and non-transgenic mice both showed significant improvements in escape latencies with time, whereas data from IgG-treated Tg2576 mice did not reach statistical significance. Between-group analysis indicated that both NAB61-treated Tg2576 and non-transgenic mice both performed significantly better than IgG-treated Tg2576 mice. (**, p < 0.01; ***, p < 0.001). D–F, spatial reference memory, percentage of time in quadrant. The percentage of time spent in each quadrant for a 60-s probe trial conducted early (D), middle (E), and late (F) during training indicated that all three groups of mice acquired some level of spatial reference memory. However, only NAB61-treated Tg2576 and non-transgenic mice spent significantly more time in the target quadrant relative to adjacent quadrants during the first probe trial (D). ns, not significant; p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. G, spatial reference memory, platform crossing index. The number of crosses over the exact location of the hidden platform subtracted by the average number of crosses over non-target platforms was measured throughout the three probe trials. NAB61-treated Tg2576 and non-transgenic mice performed significantly better than IgG-treated Tg2576 mice. (*, p < 0.05). H, motor function in the Morris water maze. All three groups of mice displayed similar swim speeds in all three probe trials.

 
Finally, passive immunization against Abeta has been postulated to enhance the efflux of Abeta from the central nervous system into the periphery. However, the presence of Abeta oligomers has not been demonstrated in the periphery, and therefore, NAB61 would not be expected to affect peripheral pools of Abeta. Immunoprecipitation of Abeta from sera obtained from passively immunized Tg2576 mice failed to demonstrate an increase in peripheral Abeta (Fig. 6D). Furthermore, naturally occurring Abeta oligomers were not immunoprecipitated from sera samples, although very low concentrations of synthetic Abeta oligomers could be immunoprecipitated when added to sera samples (Fig. 6D). Having ruled out effects on APP processing, Abeta amyloid accumulation, and peripheral pools of Abeta, the selectivity toward oligomeric Abeta exhibited by NAB61 suggested that direct neutralization of Abeta oligomers by immunization with NAB61 can reverse learning and memory deficits in Tg2576 mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite the use of a stable Abeta oligomer preparation, the generation of an anti-Abeta oligomer-selective monoclonal antibody was a low probability event, with less than 0.02% of tested hybridomas generating an antibody with selectivity toward Abeta oligomers. Indeed, although several monoclonal antibodies that recognize Abeta or nitrated epitopes were also isolated, none of these antibodies showed any Abeta oligomer selectivity. Given its unique epitope, we have thoroughly characterized NAB61 using multiple methods including immunoblotting, immunoprecipitation, solid-phase ELISA, sandwich ELISA, immunocytochemistry, immunohistochemistry, immunoelectron microscopy, and immunotherapy. In contrast with other reported oligomer- or amyloid fibril-specific antibodies (22, 23), NAB61 does not recognize other amyloidogenic proteins and is specific for Abeta, recognizing both oligomers and higher order Abeta structures. Thus, NAB61 recognized a pathologic Abeta conformation present early in the process of oligomerization, which is maintained even in Abeta fibrils. Additional polyclonal anti-Abeta sera have been reported that are apparently similar to NAB61 by immunoblotting. However, only one of the antibodies has been characterized by immunoprecipitation, immunohistochemistry, and immunoelectron microscopy (24–26). Furthermore, NAB61 is a monoclonal antibody, and therefore, suitable for passive immunization and for future development as a therapy for AD.

The occurrence of aseptic meningoencephalitis in 6% of individuals immunized with Abeta42 halted a phase II human clinical trial and calls into question the safety of active immunization for the treatment of AD (7, 8). Treatment using anti-Abeta antibodies has been proposed as a safer alternative to active immunization since immunotherapy-induced meningoencephalitis appears to be due to the activation of autoreactive T cells and does not correlate with the presence of anti-Abeta antibodies (8). Furthermore, Abeta oligomer-selective antibodies are decreased in AD patients, suggesting that replacement therapy with such antibodies may be appropriate (27). A priori, the selectivity of NAB61 for pathologic forms of Abeta, in addition to the lack of cross-reactivity with APP or C99, indicated that NAB61 may be a safer alternative for therapy when compared with other monoclonal anti-Abeta antibodies. However, we have reported one case of meningoencephalitis after peripheral immunization of a 19-month-old Tg2576 mouse with NAB61 (28). This isolated case was not part of the behavioral cohort presented here, and the mechanism triggering Abeta vaccine-related meningoencephalitis is still unknown. Nonetheless, current passive immunization trials should continue with careful regard toward unwanted complications. Additionally, the low titer of NAB61 by solid-phase ELISA indicated that the measurement of antibody response in actively immunized individuals by ELISA methods may lead to false negative results.


Figure 6
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  		FIGURE 6.
Sub-chronic NAB61 treatment does not affect APP or Abeta levels. A, APP processing in NAB61 immunized mice. Full-length APP was immunoprecipitated with a C-terminal APP antibody (5685) and immunoblotted with an N-terminal APP antibody (Karen). sAPPbetaswe levels were assessed by immunoblotting with an end-specific polyclonal antibody (54). C99 was immunoprecipitated with NAB228, run on a 10/16.5% Tris-Tricine gel, and immunoblotted with 5685. beta-tubulin was immunoblotted with TUB2.1. B, Abeta amyloid pathology in NAB61 immunized mice. Serial sections from Tg2576 mice treated with either nonspecific IgG (top) or NAB61 (bottom) were subject to immunohistochemistry using either NAB228 (left) or NAB61 (right) as primary antibodies, showing that short term NAB61 immunization did not clear amyloid deposits. C, quantification of Abeta accumulation in NAB61 immunized mice. Cortical and hippocampal regions were subject to sequential extraction with RIPA and then 70% formic acid (FA). Abeta concentrations were measured by sandwich ELISA specific for Abeta40 and Abeta42. The short NAB61 immunization protocol did not alter levels of Abeta accumulation. D, peripheral pools of Abeta in NAB61 immunized mice. Plasma samples from Tg2576 mice treated with either nonspecific IgG or NAB61 were subject to immunoprecipitation and immunoblotting with 4G8, demonstrating that NAB61 immunization does not result in the peripheral accumulation of Abeta. Although Abeta oligomers (1 ng of untreated Abeta and 1 ng of UV-cross-linked Abeta) could be detected when spiked into plasma from non-transgenic mice, no Abeta oligomers could be detected in plasma from Tg2576 mice. wt, wild type.

 
Although we have shown that NAB61 treatment improves spatial learning and memory in 17–19-month-old Tg2576 mice through the measurement of three different behavioral indices, NAB61 has been ineffective thus far in clearing Abeta pathology even in Tg2576 mice treated for 6 months. The successful clearance of amyloid pathology by passive immunization has been reported in PDAPP mice and very old (>16 months) Tg2576 mice (2, 29–32), both of which are characterized by the presence of considerable amounts of diffuse Abeta. However, our long term passive immunization trial was performed in younger Tg2576 mice (<14 months) in which compact amyloid deposits predominate, perhaps accounting for the lack of effect on the burden of Abeta pathology following the schedule used here. Alternatively, the Abeta oligomers recognized by NAB61 may not be easily cleared by immune-mediated mechanisms. NAB61 is also an IgG1 that has been shown to be less effective in reducing Abeta amyloid burden in transgenic mice (33). Indeed, distinct morphological subtypes of Abeta amyloid plaques are differentially cleared depending on the anti-Abeta antibody isotype (34). Thus, NAB61 may exert its behavioral effect by blocking the biological activity of pathologic Abeta oligomers, and at the same time, be ineffective in promoting the immune-mediated clearance of Abeta.

Finally, soluble oligomeric forms of Abeta have been postulated to contribute to the onset of AD, and they may affect neuronal function initially by impairing synaptic function (35–39). Furthermore, although insoluble amyloid plaques are found very early in the disease process in patients with early AD or mild cognitive impairment, soluble Abeta levels are also increased in these individuals, and soluble Abeta levels correlate better with neurofibrillary degeneration and the loss of synaptic markers than do amyloid plaques (40–42). Similarly, decreases in synaptophysin immunoreactivity and impairments in synaptic transmission in APP transgenic mice precede the onset of microscopic Abeta amyloid pathology (43, 44). With mounting evidence for the synaptotoxic effects of soluble Abeta oligomers, our study corroborates previous reports indicating that immunization can ameliorate cognitive defects independent of APP processing and levels of insoluble Abeta (9, 10). Furthermore, since NAB61 recognizes a conformation found on oligomeric forms of Abeta,we have provided strong evidence that Abeta oligomers generated in situ disrupt neuronal function. Thus, we propose that targeting the pathologic conformation recognized by NAB61 may be useful in the treatment of AD and that further elucidation of the conformation recognized by NAB61 may yield insights into the mechanisms underlying the synaptotoxic effects of Abeta as well as assist in determining the roles of different conformational pools of Abeta to the development of dementia.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Training Grant T32 AG00255 (to E. B. L.) and NIA, National Institutes of Health Grants AG11542 (to V. M.-Y. L.) and AG008012 (T. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains a supplemental table and supplemental methods. Back

1 The Measey-Schnabel professor of Geriatric Medicine and Gerontology. Back

2 To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, 3rd Fl., Maloney Bldg., 3600 Spruce St., Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee{at}mail.med.upenn.edu.

3 The abbreviations used are: AD, Alzheimer disease; Abeta, amyloid-beta peptide; APP, amyloid-beta precursor protein; APPswe, APP harboring the Swedish mutation; sAPPbeta, beta-cleavage-derived N-terminal ectodomain of APP; C99, beta-secretase derived C-terminal APP fragment; HNE, hydroxynonenal; MCI, mild cognitive impairment; MWM, Morris water maze; RIPA, radioimmune precipitation assay; Tricine, N-(2-hydroxyl-1,1-bis(hydroxymethyl)ethyl)glycine; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance; HRP, horseradish peroxidase; Tg, transgenic. Back


   ACKNOWLEDGMENTS
 
We gratefully thank Janssen Pharmacia and Lilly for providing monoclonal antibodies for the antibody sandwich ELISAs and H. Ischiropoulos, J. M. Souza, T. Montine, and D. Teplow for oligomeric Abeta preparations. MCI tissue sections were provided through a program project grant between Rush University and the University of Pennsylvania courtesy of E. Mufson. We also thank S. Leight and I. Solano for technical advice and assistance and A. Crystal, R. Fotina, L. Kim, K. N. Liu, J. Huse, V. Morais, and C. Wilson for valuable discussions.



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