PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2010 April 19.
Published in final edited form as:
PMCID: PMC2856604
NIHMSID: NIHMS188037

Subcutaneous Nogo Receptor Removes Brain Amyloid-β and Improves Spatial Memory in Alzheimer’s Transgenic Mice

Abstract

The production and aggregation of cerebral amyloid-β (Aβ) peptide are thought to play a causal role in Alzheimer’s disease (AD). Previously, we found that the Nogo-66 receptor (NgR) interacts physically with both Aβ and the amyloid precursor protein (APP). The inverse correlation of Aβ levels with NgR levels within the brain may reflect regulation of Aβ production and/or Aβ clearance. Here, we assess the potential therapeutic benefit of peripheral NgR-mediated Aβ clearance in APPswe/PSEN-1ΔE9 transgenic mice. Through site-directed mutagenesis, we demonstrate that the central 15–28 aa of Aβ associate with specific surface-accessible patches on the leucine-rich repeat concave side of the solenoid structure of NgR. In transgenic mice, subcutaneous NgR(310)ecto-Fc treatment reduces brain Aβ plaque load while increasing the relative levels of serum Aβ. These changes in Aβ are correlated with improved spatial memory in the radial arm water maze. The benefits of peripheral NgR administration are evident when therapy is initiated after disease onset. Thus, the peripheral association of NgR(310)ecto-Fc with central Aβ residues provides an effective therapeutic approach for AD.

Keywords: Alzheimer’s disease, β-amyloid, Nogo-66 receptor, axon, therapy, radial arm water maze, degeneration, amyloid precursor protein

Introduction

Neurodegeneration in Alzheimer’s disease (AD) is accompanied by amyloid plaques and neurofibrillary tangles (Glenner et al., 1984; Hardy and Selkoe, 2002). The amyloid plaques are composed primarily of a 40–43 aa amyloid-β (Aβ) peptide that is derived from proteolytic cleavage of amyloid precursor protein (APP) (Li et al., 1995; Sinha et al., 1999; Vassar et al., 1999). Potential therapies include decreasing β production (Lanz et al., 2003) with secretase inhibitors, increasing β degradation (Frautschy et al., 1992; Qiu et al., 1998; Bertram et al., 2000; Iwata et al., 2001; Yasojima et al., 2001), and promoting Aβ-specific immunity (Morgan et al., 2000; Lee, 2001; Younkin, 2001). However, problems with toxicity and clearing the blood–brain barrier (BBB) have hampered efforts to treat AD (Birmingham and Frantz, 2002; Orgogozo et al., 2003).

The Nogo-66 receptor (NgR) participates in limiting injury-induced axonal growth and experience-dependent plasticity in the adult brain (Fournier et al., 2001; McGee and Strittmatter, 2003; McGee et al., 2005). In this role, it serves as a receptor for three myelin inhibitor proteins, Nogo, MAG, and OMgp, signaling to activate Rho GTPase in axons (Fournier et al., 2001, 2003; Liu et al., 2002; Wang et al., 2002; McGee and Strittmatter, 2003). Recently, we found that brain NgR interacts with APP through its Aβ domain (Park et al., 2006). Moreover, increased levels of brain NgR result in reduced Aβ load, whereas loss of endogenous NgR elevates Aβ. Parallel changes in Aβ and secreted APPα plus APPβ suggest that at least a portion of the in vivo effects of brain NgR on Aβ levels is mediated by blockade of α/β-secretase activity. However, the high affinity of NgR for Aβ and the presence of NgR in plaques imply that NgR might also regulate the clearance of Aβ (Park et al., 2006).

Immunological methods have been successful in decreasing Aβ plaque burden, as reviewed by Schenk (2002). Both active and passive immunizations have promoted efflux, inhibited influx, or activated microglia-induced antibody (Ab) degradation (Schenk et al., 1999; Morgan et al., 2000; Weiner and Selkoe, 2002). Active immunization with Aβ(1–42) plus adjuvant in transgenic mutant APP reduced Aβ plaque pathology (Schenk et al., 1999). Bard et al. (2000) demonstrated that humoral immunity is sufficient to reduce plaque burden by triggering Ab trafficking across the BBB. In contrast, DeMattos et al. (2001) demonstrated that an Aβ Ab reduces Alzheimer pathology without Ab passage across the BBB, implicating a peripheral sink mechanism for anti-Aβ reductions in Alzheimer’s pathology. Different Aβ Abs recognizing various epitopes have significantly different actions in vivo. The N-terminal Aβ residues serve as robust antigens for a humoral immune response, and passive administration of some (but not all) N-terminal-directed Abs effectively remove Aβ from the brain (Bard et al., 2000). Passive transfer of one midportion Aβ Ab is highly effective in removing Aβ from the mouse brain (DeMattos et al., 2001). As antigens, the C-terminal residues of Aβ appear to yield Abs that are less effective in reducing Aβ burden (Levites et al., 2006).

In a range of studies, reducing Aβ burden in the brain by immunological means has been associated with improved spatial memory performance in Alzheimer model transgenic mice. However, in several reports, behavioral improvements occurred acutely, before any change in plaque density, suggesting the Ab association with particular soluble Aβ species is responsible for improved function. Two non-Ig proteins, RAGE and gelsolin, have been shown to bind Aβ and, when administered peripherally, to decrease brain Aβ load (Deane et al., 2003; Matsuoka et al., 2003; Arancio et al., 2004). This supports the peripheral sink hypothesis for passive immunization against Aβ. Whether Aβ reduction by peripheral non-Ab Aβ-binding proteins is associated with improved cognitive and memory function has not been tested.

Because previous studies suggest that Aβ burden can be reduced by peripheral administration of Aβ-binding agents, we hypothesized that the high affinity of NgR for Aβ should also reduce CNS Aβ levels through a peripheral sink mechanism. In this report, we define the amino acid residue requirements for the Aβ–NgR interaction. We demonstrate that subcutaneous NgR(310)ecto-Fc reduces plaque burden after disease onset in APPswe/PSEN-1ΔE9 transgenic mice. Most critically, NgR(310)ecto-Fc treatment improves short-term memory of Alzheimer transgenic mice in the radial arm water maze (RAWM) task.

Materials and Methods

NgR mutagenesis and ligand-binding assays

AP-Aβ and AP-Nogo proteins were produced as described previously (Fournier et al., 2001; Park et al., 2006). To generate AP-Aβ mutant constructs, Aβ fragments were amplified, ligated into the pAP5tag vector (GenHunter, Nashville, TN), and sequenced. Recombinant proteins were confirmed by immunoblotting. The binding of AP fusion proteins to transfected COS-7 cells has been described previously (Fournier et al., 2001). COS-7 cells were transfected with p75-NTR and RAGE, membrane proteins reported to bind Aβ (Deane et al., 2003) (Yaar et al., 1997). Synthetic Aβ(1–28) was used to assess AP-Aβ(1–28) and AP-Nogo-66(1–33) displacement from immobilized NgR(310)ecto-Fc in an ELISA format.

NgR mutagenesis has been described previously (Fournier et al., 2003; Hu et al., 2005). Bound AP was stained and measured using NIH Image software. Mutants of human NgR were detected immunofluorescently on the surface of transfected COS-7 cells. Whole COS-7 cell lysates expressing NgR mutants were subjected to SDS-PAGE and blotted with anti-NgR Abs.

Treatment of transgenic mice with NgR

To administer rat NgR(310)ecto-Fc protein, APPswe/PSEN-1ΔE9 mice (Park et al., 2006) from The Jackson Laboratory (Bar Harbor, ME) were anesthetized with isoflurane and oxygen, and an Alzet (Cupertino, CA) osmotic pump 2004 was subcutaneously inserted over the scapula and allowed to rest between fascia. The 200 µl pump delivered 0.25 µl/h for 28 d of a 1.2 µg/µl solution of rat NgR(310)Ecto-Fc or rat IgG in PBS. Pumps were replaced after 28 d for a total treatment duration of 12 weeks. The anti-Aβ (6E10) Ab was from Chemicon (Temecula, CA). DAB staining reagents were from Vector Laboratories (Burlingame, CA).

Biochemical and histological analyses

Brains from subcutaneously treated APPswe/PSEN-1ΔE9 transgenic mice were homogenized in PBS plus a protease inhibitor mixture (Roche Products, Welwyn Garden City, UK), followed by centrifugation at 100,000 × g for 20 min. Secreted APPα (sAPPα) was measured by immunoblot analysis with 6E10 anti-Aβ-(1–17) Ab (Park et al., 2006) (Chemicon). Alternatively, the brain extract was subjected to Protein A/G Plus Sepharose (Pierce, Rockford, IL) immunoprecipitation and analyzed by anti-NgR polyclonal Ab (AF1440; R & D Systems, Minneapolis, MN). Aβ ELISAs were performed according to the manufacturer’s protocol (Biosource, Camarillo, CA), as described for formic acid extracts of the brain (Park et al., 2006). Aβ plaques in parasagittal sections were fixed by paraformaldehyde and labeled with anti-Aβ-(1–17) 6E10 Ab after 0.1 m formic acid treatment (Park et al., 2006). Plaque area was quantitated as a percentage of total cerebral cortical area for two sections from each animal using NIH Image software. Neuritic dystrophy and reactive astrocytosis were visualized by staining with monoclonal anti-synaptophysin GA-5 (Sigma, St. Louis, MO) and monoclonal anti-GFAP SY38 (Chemicon) in parasagittal paraffin-embedded sections. The area of the cerebral cortex and hippocampus occupied by clusters of dystrophic neurites and reactive astrocytes were measured as a percentage of total area by the same method as Aβ plaque load (Park et al., 2006). DAB staining reagents were from Vector Laboratories.

RAWM testing

A modified RAWM testing protocol was based on a communication with D. Morgan (Wilcock et al., 2006). The maze consisted of a circular pool 1 m in diameter with six swim alleys 19 cm wide that radiated out from a 40 cm open central area, and a submerged escape platform was located at the end of one arm. Spatial cues were presented on the walls and at the end of each arm. The behaviorist was blind to the treatment and genotype. To control for vision, motivation, and swimming, mice were tested in an open-water visual-platform paradigm for up to 1 min, and latency times were recorded. Next, mice were placed in a random arm according to the following: Excel function = MOD[$CELL+RANDBETWEEN(1,5),6], where $CELL is the location of the hidden platform. Each mouse was allowed to swim for up to 1 min to find the escape platform. After entering an incorrect arm (all four paws within that swim alley) or failing to select an arm after 20 s, the mouse was pulled back to the start arm and charged an error. All mice spent 30 s on the platform after each trial before beginning the next trial. Thereafter, the mouse was tested four more times, constituting a learning block. Mice were allowed to rest for 30 min between learning blocks. In total, mice were tested over three learning blocks of five trials over the first day and then three blocks of five trials on the subsequent day. Through the group of 30 trials, the hidden platform position was kept constant for each mouse.

Statistical comparisons were made using ANOVA tests and SPSS 11 software (SPSS, Chicago, IL).

Results

Before assessing NgR as a therapeutic agent in Alzheimer model systems, we extended our characterization of the specificity of Aβ interaction with NgR. First, we sought to determine whether a linear subsegment of Aβ(1–28) might interact with NgR in a cell-binding assay. We created deletion constructs containing various portions of the Aβ ectodomain fused to AP. The region of Aβ responsible for NgR interaction localizes to residues 15–28, the central residues of Aβ(1–40) (Fig. 1a).

Figure 1
Residues 15–28 in Aβ(1–28) are essential for binding to NgR.a, Deletion mapping of the AP-Aβ(1–28) region required for binding to COS-7 cells expressing wild-type NgR. The 8–28 construct demonstrates strong ...

We also compared the binding of AP-Aβ(1–28) to NgR with other reported partners, p75 and RAGE (Yaar et al., 1997; Deane et al., 2003). Under conditions in which AP-Aβ(1–28) binding to NgR is readily detectable, p75 and RAGE do not exhibit a significant interaction with Aβ fusion protein (Fig. 1b).

NgR was identified by virtue of its affinity for Nogo-66, so we considered whether Aβ and Nogo-66 compete for binding to NgR. Competition was assessed in binding assays of AP-Aβ(1–28) or AP-Nogo66(1–33) to immobilized, purified NgR protein. Synthetic Aβ(1–28) disrupts the ability of NgR to interact with the Aβ ligand but not the Nogo-66 ligand (Fig. 1c). Thus, the two ligand-binding sites of NgR are distinguished by this assay.

To probe the NgR domains that interact with Aβ and Nogo-66, a strategy based on the crystal structure of NgR was used. A number of human NgR surface-accessible residues were mutated to Ala either individually or as groups of adjacent residues (Table 1), and resultant ligand-binding characteristics were assessed. The expression of each mutant NgR protein was verified by immunohistochemical detection at the surface of cells transfected with expression vector (Fig. 2a). For each of the mutants with altered binding characteristics, expression of immunoreactive NgR protein with electrophoretic mobility similar to wild type was also confirmed by immunoblot analysis (Fig. 2c). The mobility of each mutant was indistinguishable from wild-type NgR, except for the mutations in N-linked glycosylation sites (N82 and N179).

Figure 2
Specific residues in NgR support binding to AP-Aβ(1–28). a, Mutant NgR proteins at the surface of transfected COS-7 cells were detected by immunostaining with rabbit anti-NgR Ab recognized by anti-rabbit-AP. b, Binding of AP or AP-fused ...
Table 1
Summary of human NgR mutants: list of residues mutated to alanine

The binding of both AP-Aβ(1–28) and AP-Nogo-66 ligands to cells expressing this collection of NgR mutant proteins was assessed at concentrations equal to the predetermined KD of the ligands (Fig. 2b,d). A large group of amino acids are unnecessary for the binding of either ligand (Table 1; Fig. 2e, green); this includes all of the residues examined from the convex side of the NgR leucine-rich repeat (LRR) domain. Another subset of amino acids is essential for the binding of both Aβ(1–28) and Nogo-66 (Table 1; Fig. 2e, red). Because these amino acids do not alter the localization or molecular size of NgR protein and are clustered in close proximity on the concave surface, we hypothesize that they form a core ligand-binding site. This is consistent with the observation that for other LRR proteins, such as the follicle-stimulating hormone receptor, ligand binding predominantly occurs on the concave side (Fan and Hendrickson, 2005). Without structural studies, the possibility that these mutations prevent native NgR protein folding cannot be excluded. Most interesting are a third group of amino acids, for which Ala substitution results in NgR binding of Nogo-66 but not Aβ(1–28) (Table 1; Fig. 2e, blue or yellow). Because AP-Nogo-66 binding is indistinguishable from wild-type NgR, aberrant protein folding is unlikely to be the basis for reduced Aβ binding. Instead, NgR amino acids 210, 256, 259, and 284 are likely to contribute selectively to Aβ but not Nogo-66 interaction.

Although endogenous NgR plays a role in limiting Aβ production and deposition, the affinity of NgR for the central domain of Aβ suggests that it might promote peripheral clearance if delivered outside of the CNS. To examine whether rat NgR(310)ecto-Fc administered subcutaneously enters the brain of mouse, we assayed the presence of NgR(310)ecto-Fc in brain lysates. The NgR(310)ecto-Fc fusion protein or control rat Ig was concentrated by protein A/G affinity chromatography. Although intracerebroventricular administration leads to easily detected NgR(310)ecto-Fc levels in brain tissue, no NgR(310)ecto-Fc is detected centrally after subcutaneous treatment (Fig. 3a). This is consistent with the hypothesis that NgR(310)ecto-Fc cannot pass the BBB to an appreciable degree in APPswe/PSEN-1ΔE9 mice.

Figure 3
NgR(310)ecto-Fc treatment acts peripherally to alter the plasma/brain Aβ ratio. a, An anti-NgR immunoblot of protein A/G binding proteins in brain lysate of APPswe/PSEN-1ΔE9 transgenic mice treated subcutaneously (sc) with rat IgG, subcutaneously ...

To the extent that NgR(310)ecto-Fc functions as a peripheral sink for Aβ, the ratio of plasma to brain Aβ should be elevated, as shown for anti-Aβ treatment. Levels of Aβ40 and Aβ42 were assessed by ELISA in brain and plasma samples from peripherally treated mice (Fig. 3b). Subcutaneous treatment with NgR(310)ecto-Fc increases plasma to brain ratios for Aβ by more than twofold. The increase in this ratio is primarily attributable to a decrease in brain levels with NgR(310)ecto-Fc treatment (see brain analysis below) and not to a change in plasma levels (Fig. 3c). Previously, we noted that central, intracerebroventricularly administered NgR(310)ecto-Fc reduces levels of sAPPα and sAPPβ protein in the brain (Park et al., 2006). If subcutaneous NgR(310)ecto-Fc acts to alter Aβ clearance in the periphery, and not to alter Aβ production centrally, then similar changes in APP should fail to occur with peripheral treatment. Indeed, brain APP levels are not altered by subcutaneous NgR(310)ecto-Fc (Fig. 3d,e).

The restriction of subcutaneous NgR(310)ecto-Fc to the periphery allows an assessment of its effect as a “sink” on central Aβ burden. Treatment of APPswe/PS-1ΔE9 transgenic mice was initiated at 7 months of age when the mice have become symptomatic, as judged by Aβ deposition in brain and by reduced spatial memory function (see below). After 3 months of subcutaneous treatment with 0.27 mg/kg/d NgR(310)ecto-Fc versus IgG, the brain was examined. The total Aβ(1–40) and Aβ(1–42) levels as well as Aβ plaque are decreased significantly by NgR(310)ecto-Fc, to a level ~50% less than the control (Fig. 4a,d,e). In parallel, dystrophic neurites detected by antisynaptophysin staining are decreased by peripheral NgR(310)ecto-Fc treatment (Fig. 4b,f). Astrogliosis detected by anti-GFAP staining intensity was also reduced significantly by therapy with peripheral NgR(310)ecto-Fc (Fig. 4c,g). Thus, delayed subcutaneous administration of NgR(310)ecto-Fc suppresses histologic evidence of Aβ-associated disease in transgenic mice.

Figure 4
Reduction in Aβ plaque load, neuritic dystrophy, and astrocytosis in NgR(310)ecto-Fc-treated APPswe/PSEN-1ΔE9 mice. Sections of cerebral cortex from APPswe/PSEN-1ΔE9 transgenic mice treated with rat IgG or NgR(310)ecto-Fc are shown. ...

The ability of subcutaneous NgR(310)ecto-Fc therapy to reduce Aβ plaque is encouraging, but cognitive performance is the relevant symptom in clinical AD. To assess APPswe/PSEN-1ΔE9 transgene-related impairments in spatial memory and learning over a 24 h period, we used a modified RAWM paradigm (Wilcock et al., 2006). If performance on this task reflects spatial learning and not alterations in motivation, motor coordination, stamina or vision, then the latency to reach a visible escape platform should not differ between groups. Such latencies are indistinguishable between littermate-matched wild-type and APPswe/ PS-1ΔE9 mice (38 ± 2 s vs 42 ± 5 s, respectively; mean ± SEM; n = 8–10 mice). Learning deficits in the 24 h spatial memory task are apparent in APPswe/PSEN-1ΔE9 versus wild-type littermate mice by 4 months of age (Fig. 5a). By 13 months of age, wild-type mice perform less well at this task than do young mice, whereas APPswe/PSEN-1ΔE9 transgenic mice are completely unable to learn the task in our training paradigm illustrating disease progression (Fig. 5b). As a control, loss of NgR expression (in ngr−/− mice) does not significantly alter RAWM performance (Fig. 5c).

Figure 5
Subcutaneous treatment of NgR(310)ecto-Fc improves RAWM performance in APPswe/PSEN-1ΔE9 transgenic mice.a, Average number of errors for the indicated swim trials in a six-arm radial water mazeforAPPswe/PSEN-1ΔE9 and wild-type littermates ...

The number of swim errors made by APPswe/PS-1ΔE9 mice after 20 training trials increases steadily at 8, 9, and 10 months when mice receive control IgG therapy subcutaneously for 1, 2, or 3 months. In contrast, mice treated with subcutaneous NgR(310)ecto-Fc exhibit a halt in disease progression and show a trend toward improved performance after 3 months, by 10 months of age (Fig. 5d). RAWM errors are significantly reduced after 2 and 3 months of subcutaneous NgR(310)ecto-Fc treatment compared with rat IgG-treated mice (ANOVA, p < 0.05 and 0.02, respectively). These differences are related to improved memory function rather than altered vision, motivation, or motor capacity because no significant difference was observed in visible platform escape latencies between these groups (supplemental Fig.1, available at www.jneurosci.org as supplemental material). The improved performance of the NgR(310)ecto-Fc-treated mice is not present during initial swim trials but develops during the 24 h training period (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). There is a positive correlation between the average RAWM errors and the density of Aβ-immunoreactive deposits across the two groups (Fig. 5e).

Discussion

In the present study, we have defined the molecular requirements for Aβ interaction with NgR and exploited the affinity of these polypeptides to demonstrate a subcutaneous method for alleviating memory impairments in AD model mice. The 15–28 central residues of Aβ bind to a pocket on the concave surface of NgR that can be distinguished from the surface required for Nogo-66 binding. When NgR is administered subcutaneously, the protein does not enter the brain but can functionally shift Aβ from the brain to plasma. The net result of this shift is a lessening of AD-related pathology. Most critically, peripheral NgR(310)ecto-Fc improves memory performance even when treatment is initiated after symptoms develop. Thus, NgR therapy provides an alternative to immune-based Aβ therapies for AD.

In previous work, we had administered NgR(310)ecto-Fc intracerebroventricularly rather than subcutaneously to transgenic AD mice (Park et al., 2006). Similar reductions in Aβ were detected, but memory deficits were not assessed. Interestingly, the central administration of NgR(310)ecto-Fc reduced brain sAPPα as well as Aβ. Furthermore, the elevated Aβ and sAPPa in mice lacking NgR indicated that endogenous neuronal NgR serves to limit Aβ production. Thus, the binding of excess NgR to both APP and Aβ has the potential to reduce Aβ production as well as to enhance Aβ clearance. The predominant action of subcutaneous NgR is the acceleration of Aβ clearance.

The reduction in both Aβ and memory deficits with a nonimmunologic NgR protein provides additional evidence for the amyloid hypothesis of AD and also for the peripheral sink hypothesis of anti-Aβ Ab function. Several previous studies demonstrate that peripheral administration of Abs directed against Aβ reduces AD pathology. The Aβ residues recognized by NgR match most closely to the Aβ(13–28) epitope reacting with the m266 Ab. Like NgR(310)ecto-Fc, the m266 Ab reduces plaque burden (DeMattos et al., 2001; Dodart et al., 2002). As opposed to active or passive immunologic reagents, NgR is an endogenous protein and is not predicted to activate or to require cell-mediated mechanisms to reduce Aβ levels and Aβ-associated cognitive deficits. Two other non-Ig proteins, RAGE and gelsolin, have been shown to bind Aβ and to decrease brain Aβ load (Deane et al., 2003; Matsuoka et al., 2003; Arancio et al., 2004) when administered peripherally. However, the effect of these proteins on memory function has not been examined, and shifts in Aβ are complicated by the fact that endogenous gelsolin is cytoplasmic and that RAGE alters BBB transport (Deane et al., 2003).

Several recent studies indicate that specific Aβ isoforms, such as soluble oligomers including Aβ*56, are better correlated with memory impairment than the total Aβ load in the brain (McLaurin et al., 2006). In our study, errors in RAWM performance and total brain Aβ deposition are directly correlated. This suggests that NgR(310)ecto-Fc does not distinguish between Aβ isoforms. The time course of improvement in memory performance after NgR(310)ecto-Fc is slow. After 1 month, the differences are minimal, whereas they are clear at 2 months and dramatic at 3 months of treatment. This suggests that NgR is not binding acutely to a small pool of a function-blocking species of Aβ to promote rapid reversal of cognitive impairment but, rather, is leading to a shift in the steady-state levels of multiple Aβ forms in the brain through slow equilibration with species bound peripherally to the infused NgR protein. Future studies are required to directly assess NgR affinity for different Aβ forms and their regulation by subcutaneous NgR(310)ecto-Fc infusion.

In the current study, NgR affinity for Aβ has been defined and exploited for therapeutic benefit. CNS myelin also contains ligands for the NgR. The interaction of myelin with NgR limits experience-dependent plasticity in the cerebral cortex (McGee et al., 2005) and injury-induced regenerative axonal growth (GrandPre et al., 2002; Li and Strittmatter, 2003; Kim et al., 2004; Li et al., 2004, 2005). The influence of Aβ with the myelin/NgR within the brain remains to be defined. Because human Aβ binding to human NgR exhibits a substantially higher affinity than mouse Aβ for mouse NgR (Park et al., 2006), and because Aβ levels are low without disease, the Aβ–NgR interaction may function only in the pathological AD state. Alternatively, Aβ regulation of NgR-dependent axonal plasticity may contribute to impaired cognitive function in AD.

The LRR protein family is involved in multiple protein–protein interactions. LRR domains contribute to the ectodomain segment of a range of cell-surface receptors, including NgR, Trk receptors, Toll-like receptors, follicle-stimulating hormone/luteinizing hormone and related G-protein-coupled receptors, and platelet GP1bα (Kobe and Deisenhofer, 1994). Like the NgR, the platelet GP1bα interacts with unrelated protein ligands, von Willebrand’s factor, and thrombin. Although Aβ, Nogo-66, MAG, and OMgp share no sequence similarity, they each bind to NgR. For Aβ and Nogo-66, binding sites defined by mutagenesis appear closely apposed along the midportion of the concave surface. Structural studies of ligand/receptor will be required to further illuminate NgR binding and to explore the possibility of small molecule NgR ligand compounds as therapeutic agents. In summary, we show that subcutaneous NgR(310)ecto-Fc binds Aβ in the periphery to reduce Aβ and improve spatial memory in a mouse model of AD.

Supplementary Material

Supplemental Figure S1.

Supplemental Figure S2.

Acknowledgments

This work was supported by grants from the National Institutes of Health (NIH) to S.M.S. and by an institutional NIH Medical Scientist Training grant to J.H.P. S.M.S. is a member of the Kavli Institute of Neuroscience at Yale University. We thank Dave Morgan for counsel regarding radial arm water maze testing.

References

  • Arancio O, Zhang HP, Chen X, Lin C, Trinchese F, Puzzo D, Liu S, Hegde A, Yan SF, Stern A, Luddy JS, Lue LF, Walker DG, Roher A, Buttini M, Mucke L, Li W, Schmidt AM, Kindy M, Hyslop PA, et al. RAGE potentiates Abeta-induced perturbation of neuronal function in transgenic mice. EMBO J. 2004;23:4096–4105. [PubMed]
  • Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Lieberburg I, Motter R, Nguyen M, Soriano F, Vasquez N, Weiss K, Welch B, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 2000;6:916–919. [PubMed]
  • Bertram L, Blacker D, Mullin K, Keeney D, Jones J, Basu S, Yhu S, McInnis MG, Go RC, Vekrellis K, Selkoe DJ, Saunders AJ, Tanzi RE. Evidence for genetic linkage of Alzheimer’s disease to chromosome 10q. Science. 2000;290:2302–2303. [PubMed]
  • Birmingham K, Frantz S. Set back to Alzheimer vaccine studies. Nat Med. 2002;8:199–200. [PubMed]
  • Deane R, Du Yan S, Submamaryan RK, LaRue B, Jovanovic S, Hogg E, Welch D, Manness L, Lin C, Yu J, Zhu H, Ghiso J, Frangione B, Stern A, Schmidt AM, Armstrong DL, Arnold B, Liliensiek B, Nawroth P, Hofman F, et al. RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat Med. 2003;9:907–913. [PubMed]
  • DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA. 2001;98:8850–8855. [PubMed]
  • Dodart JC, Bales KR, Gannon KS, Greene SJ, DeMattos RB, Mathis C, De-Long CA, Wu S, Wu X, Holtzman DM, Paul SM. Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer’s disease model. Nat Neurosci. 2002;5:452–457. [PubMed]
  • Fan QR, Hendrickson WA. Structure of human follicle-stimulating hormone in complex with its receptor. Nature. 2005;433:269–277. [PubMed]
  • Fournier AE, GrandPre T, Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature. 2001;409:341–346. [PubMed]
  • Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neurosci. 2003;23:1416–1423. [PubMed]
  • Frautschy SA, Cole GM, Baird A. Phagocytosis and deposition of vascular beta-amyloid in rat brains injected with Alzheimer beta-amyloid. Am J Pathol. 1992;140:1389–1399. [PubMed]
  • Glenner GG, Wong CW, Quaranta V, Eanes ED. The amyloid deposits in Alzheimer’s disease: their nature and pathogenesis. Appl Pathol. 1984;2:357–369. [PubMed]
  • GrandPre T, Li S, Strittmatter SM. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature. 2002;417:547–551. [PubMed]
  • Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002;297:353–356. [PubMed]
  • Hu F, Liu BP, Budel S, Liao J, Chin J, Fournier A, Strittmatter SM. Nogo-A interacts with the Nogo-66 receptor through multiple sites to create an isoform-selective subnanomolar agonist. J Neurosci. 2005;25:5298–5304. [PMC free article] [PubMed]
  • Iwata N, Tsubuki S, Takaki Y, Shirotani K, Lu B, Gerard NP, Gerard C, Hama E, Lee HJ, Saido TC. Metabolic regulation of brain Abeta by neprilysin. Science. 2001;292:1550–1552. [PubMed]
  • Kim JE, Liu BP, Park JH, Strittmatter SM. Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury. Neuron. 2004;44:439–451. [PubMed]
  • Kobe B, Deisenhofer The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci. 1994;19:415–421. [PubMed]
  • Lanz TA, Himes CS, Pallante G, Adams L, Yamazaki S, Amore B, Merchant KM. The gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester reduces A beta levels in vivo in plasma and cerebrospinal fluid in young (plaque-free) and aged (plaque-bearing) Tg2576 mice. J Pharmacol Exp Ther. 2003;305:864–871. [PubMed]
  • Lee VM. Abeta immunization: moving Abeta peptide from brain to blood. Proc Natl Acad Sci USA. 2001;98:8931–8932. [PubMed]
  • Levites Y, Das P, Price RW, Rochette MJ, Kostura LA, McGowan EM, Murphy MP, Golde TE. Anti-Abeta42- and anti-Abeta40-specific mAbs attenuate amyloid deposition in an Alzheimer disease mouse model. J Clin Invest. 2006;116:193–201. [PMC free article] [PubMed]
  • Li J, Ma J, Potter H. Identification and expression analysis of a potential familial Alzheimer disease gene on chromosome 1 related to AD3. Proc Natl Acad Sci USA. 1995;92:12180–12184. [PubMed]
  • Li S, Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci. 2003;23:4219–4227. [PubMed]
  • Li S, Liu BP, Budel S, Li M, Ji B, Walus L, Li W, Jirik A, Rabacchi S, Choi E, Worley D, Sah DW, Pepinsky B, Lee D, Relton J, Strittmatter SM. Blockade of nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble nogo-66 receptor promotes axonal sprouting and recovery after spinal injury. J Neurosci. 2004;24:10511–10520. [PubMed]
  • Li S, Kim JE, Budel S, Hampton TG, Strittmatter SM. Transgenic inhibition of Nogo-66 receptor function allows axonal sprouting and improved locomotion after spinal injury. Mol Cell Neurosci. 2005;29:26–39. [PMC free article] [PubMed]
  • Liu BP, Fournier A, GrandPre T, Strittmatter SM. Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science. 2002;297:1190–1193. [PubMed]
  • Matsuoka Y, Saito M, LaFrancois J, Saito M, Gaynor K, Olm V, Wang L, Casey E, Lu Y, Shiratori C, Lemere C, Duff K. Novel therapeutic approach for the treatment of Alzheimer’s disease by peripheral administration of agents with an affinity to β-amyloid. J Neurosci. 2003;23:29–33. [PubMed]
  • McGee AW, Strittmatter SM. The Nogo-66 receptor: focusing myelin inhibition of axon regeneration. Trends Neurosci. 2003;26:193–198. [PubMed]
  • McGee AW, Yang Y, Fischer QS, Daw NW, Strittmatter SM. Experience-driven plasticity of visual cortex limited by myelin and Nogo receptor. Science. 2005;309:2222–2226. [PMC free article] [PubMed]
  • McLaurin J, Kierstead ME, Brown ME, Hawkes CA, Lambermon MH, Phinney AL, Darabie AA, Cousins JE, French JE, Lan MF, Chen F, Wong SS, Mount HT, Fraser PE, Westaway D, George-Hyslop PS. Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse Alzheimer phenotype in a mouse model. Nat Med. 2006;12:801–808. [PubMed]
  • Morgan D, Diamond DM, Gottschall PE, Ugen KE, Dickey C, Hardy J, Duff K, Jantzen P, DiCarlo G, Wilcock D, Connor K, Hatcher J, Hope C, Gordon M, Arendash GW. Abeta peptide vaccination prevents memory loss in an animal model of Alzheimer’s disease. Nature. 2000;408:982–985. [PubMed]
  • Orgogozo JM, Gilman S, Dartigues JF, Laurent B, Puel M, Kirby LC, Jouanny P, Dubois B, Eisner L, Flitman S, Michel BF, Boada M, Frank A, Hock C. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology. 2003;61:46–54. [PubMed]
  • Park JH, Gimbel DA, GrandPre T, Lee JK, Kim JE, Li W, Lee DH, Strittmatter SM. Alzheimer precursor protein interaction with the Nogo-66 receptor reduces amyloid-β plaque deposition. J Neurosci. 2006;26:1386–1395. [PMC free article] [PubMed]
  • Qiu WQ, Walsh DM, Ye Z, Vekrellis K, Zhang J, Podlisny MB, Rosner MR, Safavi A, Hersh LB, Selkoe DJ. Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J Biol Chem. 1998;273:32730–32738. [PubMed]
  • Schenk D. Amyloid-beta immunotherapy for Alzheimer’s disease: the end of the beginning. Nat Rev Neurosci. 2002;3:824–828. [PubMed]
  • Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, et al. Immunization with amyloid-beta attenuates Alzheimer-diseaselike pathology in the PDAPP mouse. Nature. 1999;400:173–177. [PubMed]
  • Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan M, Dovey HF, Frigon N, Hong J, Jacobson-Croak K, Jewett N, Keim P, Knops J, Lieberburg I, Power M, Tan H, Tatsuno G, Tung J, Schenk D, et al. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature. 1999;402:537–540. [PubMed]
  • Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis JC, et al. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 1999;286:735–741. [PubMed]
  • Wang KC, Koprivica V, Kim JA, Sivasankaran R, Guo Y, Neve RL, He Z. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature. 2002;417:941–944. [PubMed]
  • Weiner HL, Selkoe DJ. Inflammation and therapeutic vaccination in CNS diseases. Nature. 2002;420:879–884. [PubMed]
  • Wilcock DM, Alamed J, Goltschall PE, Grimm J, Rosenthal A, Pons J, Ronan V, Symmonds K, Gordon MN, Morgan D. Deglycosylated anti-amyloidβ antibodies eliminate cognitive deficits and reduce parenchymal amyloid with minimal vascular consequences in aged amyloid precursor protein transgenic mice. J Neurosci. 2006;26:5340–5346. [PubMed]
  • Yaar M, Zhai S, Pilch PF, Doyle SM, Eisenhauer PB, Fine RE, Gilchrest BA. Binding of beta-amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer’s disease. J Clin Invest. 1997;100:2333–2340. [PMC free article] [PubMed]
  • Yasojima K, Akiyama H, McGeer EG, McGeer PL. Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide. Neurosci Lett. 2001;297:97–100. [PubMed]
  • Younkin SG. Amyloid beta vaccination: reduced plaques and improved cognition. Nat Med. 2001;7:18–19. [PubMed]