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Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ~70% of amyloid β-peptide (Aβ) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aβ peripheral binding and higher levels of free Aβ in plasma. Experimental studies have shown that free circulating Aβ re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aβ from entering the brain. Treatment of Alzheimer APPsw+/0 mice with WT-LRPIV has been shown to reduce brain Aβ pathology. In addition to Aβ, LRPIV binds multiple ligands. To enhance LRPIV binding for Aβ relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aβ40 and Aβ42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3–1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aβ40 and Aβ42 25–27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw+/0 mice with LRPIV-D3674G (40 μg/kg/day) reduced Aβ40 and Αβ42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60–80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aβ clearance therapy.
Deposits of amyloid β-peptide (Aβ)3 and neurofibrillary tangles are neuropathological features of Alzheimer disease (AD) (1). Aβ is produced in the brain and periphery by proteolytic cleavage from its larger Aβ precursor protein (APP) (2). A body of evidence suggests that soluble Aβ oligomer species contribute to neurodegeneration in AD (3). Aβ concentration in brain interstitial fluid is controlled by its rate of production in brain; influx and/or re-entry of circulating, peripheral Aβ into the brain across the blood-brain barrier (BBB) via receptor for advanced glycation end products (RAGE) (4); and clearance of Aβ from the brain (5, 6). Aβ is cleared from the brain by different mechanisms, including transport across the BBB via low density lipoprotein receptor-related protein-1 (LRP1) (5, 7–9) and enzymatic degradation in the brain (10). A continuous removal of Aβ from the brain and systemic circulation is essential to prevent accumulation of toxic soluble oligomeric Aβ in the brain (3, 11).
We and others have reported that cell surface LRP1 at the BBB and soluble LRP1 (sLRP1) in plasma have important functions in Aβ homeostasis (7–9, 12–21). sLRP1 is a major transport binding protein for peripheral circulating Aβ and can sequester 70–90% of plasma Aβ in neurologically intact human subjects (14). sLRP1 levels are reduced in patients with AD and mild cognitive impairment who subsequently progress to AD (MCI-AD) (14, 22). In addition, sLRP1 is oxidized in AD and AD-MCI individuals, and oxidized sLRP1 is unable to bind Aβ (14, 22). Consequently, an increase in free, unbound Aβ plasma levels relative to sLRP1-bound plasma Aβ levels has been reported in AD and MCI-AD patients (14, 22). Experimental studies have shown that free Aβ40 and Aβ42 re-enter the brain (4, 23, 24) and contribute to the formation of neurotoxic soluble oligomeric Aβ species. It has been also reported that sLRP1 and its recombinant ligand binding domain cluster IV (LRPIV) prevent free Aβ from entering the brain (14).
Several Aβ antibody therapies are directed at facilitating Aβ clearance from the brain (25, 26). Aβ antibodies that act principally by sequestering the peripheral Aβ pool do not cross the BBB and are generally thought to clear Aβ from the brain by the so-called “peripheral sink” mechanism through binding of peripheral Aβ, which according to some studies lessens the risk of potential central side effects of the antibody clearance therapy, such as neuroinflammation, vasogenic edema, and cerebral microhemorrhages (27–30). Recent studies in patients with mild AD have shown beneficial effects of intravenous immunoglobulin preparation (Gammagard), which has been suggested to act as a peripheral sink agent containing sLRP1 and antibodies against diverse Aβ conformations (31, 32).
In a previous study, we have demonstrated that that wild type recombinant LRPIV (WT-LRPIV) effectively binds Aβ40 and Aβ42 in vitro (8, 14) and sequesters free Aβ in plasma of AD patients and AD transgenic mice in vivo, which reduces Aβ pathology in mice (14). LRPIV contains 11 complement-type repeats (CRs) (CR21–CR31), which can participate in binding of LRP1 ligands (33, 34). For example, CR24–CR28 efficiently binds LRP1 ligands Factor IXa, receptor-associated protein (RAP), and activated α2-macroglobulin (α2M*) (35, 36). Each CR has ~40 amino acids and a single calcium ion (35). Calcium ion binding domains are required for proper folding and structural integrity of LRP1 (33, 34). The alterations in calcium binding sites alter folding of the CRs (33). In an attempt to enhance Aβ binding to LRPIV and improve its Aβ-clearing properties, we generated a library of recombinant LRPIV fragments and full-length LRPIV variants with single glycine replacement with an aspartic acid residue in the calcium binding site in either CR 21, 22, 26, 27, 28, or 29. We expressed these LRPIV analogs in Chinese hamster ovary (CHO) cells. Next, LRP-IV-derived analogs that have been well secreted by CHO cells into the medium were purified, and their binding affinity for Aβ40 and Aβ42 and other LRP1 ligands, including apolipoprotein E2-4 (apoE2-4), tissue plasminogen activator (tPA), matrix metalloproteinase-9 (MMP-9), and Factor IXa, was determined. A lead LRPIV-D3674G mutant had the highest in vitro binding affinity for Aβ peptides relative to other ligands and cleared mouse endogenous Aβ more efficiently than WT-LRPIV. Moreover, subcutaneous LRPIV-D3674G treatment significantly reduced Aβ levels in brain and cerebrospinal fluid (CSF) in Alzheimer APPsw+/0 mice and improved cerebral blood flow responses and hippocampal function. Our findings suggest that LRPIV-D3674G is an efficient new Aβ clearance therapy for AD.
Human Aβ40 and Aβ42 were synthesized at the W. M. Keck Facility (Yale University), using solid-phase N-t-butyloxycarbonyl chemistry, and purified by HPLC. Primers were synthesized by Integrated DNA Technologies (Coralville, IA), and dNTPs were obtained from Invitrogen. All other reagents were from Sigma-Aldrich unless otherwise indicated. Purified recombinant full-length wild type ligand binding cluster IV of LRP1 (WT-LRPIV) was used for generating rabbit polyclonal LRPIV antibody (GeneScript, Piscataway, NJ). Another rabbit polyclonal anti-LRPIV antibody (37) was kindly provided by Dr. Elizabeth Komives (University of California San Diego, La Jolla, CA).
LRP1 cDNA was synthesized from human spleen total RNA (Clontech, Mountain View, CA) using SuperScript II RT (Invitrogen). Primers were designed based on LRP1 sequence (NM_002332) (Table 1). LRPIV (Fig. 1A) was amplified from cDNA by polymerase chain reaction (PCR) using Pfx-DNA polymerase (Invitrogen) and their respective primer sets and cloned into pcDNA3.3 TOPO vector. Using this construct, full-length LRPIV consisting of 11 CRs (CR21–CR31), two four-repeat fragments (CR24–CR27 and CR25–CR28), one three-repeat fragment (CR25–CR27), two two-repeat fragments (CR25-CR26 and CR26-CR27), and two single-repeat fragments (CR25 and CR26) (Fig. 1A) were amplified using PCR and cloned in mammalian expression vector pSecTag2 B (Invitrogen) between HindIII and BamHI restriction sites to express soluble proteins. pSecTag2 B vector has the IgK leader peptide on the N terminus and a Myc tag and His6 tag on the C terminus. In addition, a full-length LRPIV was amplified using 129 bp of forward primer (which has Kozak sequence, start codon, tPA signal peptide sequence, and LRPIV sequence) and reverse primer with the HindIII restriction site and cloned into pcDNA3.3 TOPO vector (WT-LRPIV). Full-length LRPIV variants with glycine replacing aspartic acid residues 3354, 3394, 3556, 3595, 3633, and 3674 (Fig. 1B) in the calcium binding sites were made by site-directed mutagenesis using the QuikChange Lightning site-directed mutagenesis kit (Stratagene, LA Jolla, CA). WT-LRPIV was used as a template along with their respective primer sets.
CHO cells were grown in CDOpti CHO medium supplemented with 1 mm CaCl2 and 2 mm Glutamax at 37 °C in a shaker flask. The cells were then stably transfected with each construct using FreeStyle MAX reagent (Invitrogen). Five days after transfection, cells were grown into medium supplemented with 700 μg/ml Geneticin (for selection of cells with pcDNA 3.3 TOPO vector) or 200 μg/ml hygromycin (for selection of cells with pSecTag2 vector). After 12–15 days, around 5000 antibiotic-resistant cells were plated on a 100 × 10-mm Petri plate containing CloneMatrix (catalog no. K8510, Genetix Molecular Devices, Inc., Sunnyvale, CA) mixture (40% CloneMatrix, 50% 2× CDOpti CHO, and antibiotics). Three weeks after plating, 50–60 single clones were transferred into CDOpti CHO medium in 48-well plates. Three days later, media were collected and tested for expression of LRPIV by Western blot analysis. Selected clones were subsequently transferred into 12-well plates. A single selected clone was transferred into a Fernbach flask and grown as a suspension culture. Culture was started with 1 × 106/ml cell density in CDOpti CHO medium containing 2 mm Glutamax, 1 mm CaCl2, and 10% CHO CD Efficient Feed A (Invitrogen). The cells were counted daily using a hemocytometer (Hausser Scientific Partnership, Horsham, PA), and glucose levels were determined using a GlucCellTM test strip (CESCO Bioengineering Co., Taichung, Taiwan). When the glucose level fell below 2 g/liter in the conditioned medium, cells were supplemented with 10% Feed A containing 2 mm Glutamax and 1 mm CaCl2. After 10 days of culture, the conditioned medium was collected, centrifuged, filtered through 0.2-μm membrane, and stored frozen at −20 °C until analysis. The LRPIV-derived peptides CR25-CR26, CR26-CR27, CR25, CR26, and mutant proteins D3354G, D3595G, and D3633G were not included in the present study due to their poor secretion in the medium.
Full-length LRPIV (CR21–CR31) and LRPIV CRs containing His6 tag were purified using Ni2+-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA). Briefly, conditioned medium was mixed with 10% glycerol, 150 mm NaCl, 10 mm imidazole, and washed Ni2+-nitrilotriacetic acid resin, rocked at room temperature for 30 min, and washed with a buffer (10% glycerol, 300 mm NaCl, 10 mm imidazole, and 50 mm NaH2PO4, pH 8). Bound protein was eluted with 250 mm imidazole in 50 mm phosphate buffer, pH 8. Eluted protein was passed through a 50-kDa cut-off filter (Millipore, Billerica, MA). The WT-LRPIV was purified by affinity purification using a GST-RAP affinity column as described (38). GST-RAP was expressed as reported (39) and affinity-purified using the B-PER GST fusion protein purification kit (Pierce). The GST-RAP affinity column was prepared using the AminoLink Plus immobilization kit (catalog no. 44894, Pierce). Mutant variants of LRPIV were purified using an anti-LRPIV-antibody affinity column. The anti-LRPIV-antibody column was prepared by immobilizing purified anti-LRPIV antibody (kindly provided by Dr. Komives) using the AminoLink Plus immobilization kit (Pierce). For purification of full-length LRPIV variants, about 100 ml of conditioned medium was diluted 3-fold with a wash buffer (20 mm Tris, 150 mm NaCl, pH 7.4) and loaded onto an anti-LRPIV antibody affinity column. The column was washed with 900 ml of wash buffer, and the bound protein was eluted with 0.1 m glycine buffer (pH 2.5), neutralized with 2 m Tris buffer (pH 9.5), and concentrated using a 10-kDa cut-off filter (Millipore). Each purified LRPIV variant was dialyzed against 50 mm carbonate-bicarbonate buffer (pH 9), and their purity was confirmed by silver staining (Fig. 1C). We screened eight LRPIV-derived peptides/proteins, which include LRPIV CRs (CR21–CR31, CR24–CR27, CR25–CR28, and CR25–CR27), WT-LRPIV, and full-length LRPIV variants (LRPIV-D3394G, LRPIV-D3556G, and LRPIV-D3674G) for their binding to Aβ40 and Aβ42 by ELISA.
Microtiter plates were coated with 5 μg/ml LRPIV analog overnight in 55 mm sodium bicarbonate buffer at 4 °C. All wells were blocked with protein-free buffer (catalog no. 37570, Pierce) for 1 h at room temperature. Varying concentrations of Aβ40, Aβ42, or ApoE2, -3, or -4 (catalog nos. P2002, P2003, and P2004, respectively, Invitrogen), tPA (catalog no. 10-633-45291, Genway Biotech, Inc., San Diego, CA), MMP-9 (catalog no. 911-MP-010, R&D Systems, Minneapolis, MN), and Factor IXa (catalog no. RP-43110, Pierce) were incubated in Hanks' balanced solution culture medium (HBSC), pH 7.4, containing 0.05% Tween 20 (HBSCT) at room temperature for 2 h. Anti-Aβ (1 μg/ml; catalog no. 2454, Cell Signaling Technology Inc.), anti-ApoE (0.1 μg/ml; catalog no. K74180, Biodesign Meridian LifeScience, Memphis, TN), anti-tPA (catalog no. Ab62763, Abcam, Cambridge, MA), anti-MMP-9 (catalog no. Ab5707, Abcam), or anti-Factor IXa (catalog no. LS-C23381, LifeSpan Biosciences, Inc., Seattle, WA) overnight at 4 °C in HBSCT containing 0.25% BSA. After washing plates four times with HBSCT, wells were incubated with goat anti rabbit-HRP or donkey anti-goat (1:3000 dilution) in HBSCT and 0.25% BSA for 30 min at room temperature. After washing plates four times with HBSCT, 100 μl of 3,3′,5,5′-tetramethylbenzidine substrate (catalog no. 53-00-01, KPL, Gaithersburg, MD) was added. The reaction was developed for 10 min and stopped with 100 μl of 1 m HCl. The absorbance was read at 450 nm. To calculate the Kd (ligand concentration that binds to half of the microtiter plate immobilized LRPIV receptor sites at equilibrium), we utilized GraphPad Prism software (version 3) based on the Marquardt method of nonlinear one-site binding (hyperbola) regression (curve fit) analysis of the ELISA-based absorbance values against specific concentrations of the various ligands studied. GraphPad Prism software uses the following equation to calculate Kd: Y = Bmax × X/(Kd + X), where Bmax is the maximum specific binding in the same units as Y (Y is the specific binding extrapolated to very high concentrations of ligand, so its value is almost always higher than any specific binding measured in the experiment). Kd is the equilibrium binding constant, in the same units as X (X is the ligand concentration needed to achieve a half-maximum binding at equilibrium). We only measured LRP-bound ligand for the affinity calculation and did not measure free and unbound LRP constructs.
The ELISA plates were coated with 10 μg/ml LRPIV-D3674G, and the plates were blocked with protein-free blocking buffer (catalog no. 37570, Pierce) as described (40). Aβ40 (100 nm) was prepared in HBSC, pH 7.4, for 2 h at room temperature. After washing with HBSC containing 0.05% Tween 20, an Aβ N terminus-specific antibody (1 μg/ml; catalog no. 2454, Cell Signaling), HRP-conjugated C terminus-specific antibody (BA27, WAKO ELISA kit), or non-immune IgG (NI-IgG) primary antibody was added and incubated overnight. The secondary detection antibody for the N terminus anti-Aβ antibody was goat anti-rabbit (1:2000 dilution; Dako). The reaction was developed with 3,3′,5,5′ tetramethylbenzidine (KPL) and stopped with 1 m HCl. Absorbance was read at 450 nm (40).
Oligomers and fibrils of Aβ40 were prepared as described earlier (41, 42) and confirmed by a dot blot assay using oligomer- and fibril-specific antibodies A11 and OC, respectively (41, 42). In the dot blot assay, 1 μg of Aβ40 monomers, oligomers, or fibrils was applied to a nitrocellulose membrane, and the membrane was blocked with protein-free blocking buffer (Pierce). The membrane was incubated with 10 μg/ml LRPIV-D3674G in HBSC, pH 7.4, for 2 h at room temperature, and the membrane was probed with a rabbit polyclonal anti-LRPIV antibody.
C57Bl6 and APPsw+/0 mice were purchased from Jackson Laboratories and Taconic Farms, respectively. Mice were housed under standard conditions (12-h light/dark cycle starting at 7:00 a.m.; 21 ± 2 °C; 55 ± 10% humidity) in solid bottomed cages on wood chip bedding. All studies were performed according to National Institutes of Health guidance using protocols approved by the University of Rochester Committee on Animal Resources. Mice were anesthetized by intraperitoneal injections with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg).
WT-LRP-IV and LRPIV-D3674G were radiolabeled with 125I by the Pierce IODO-GEN® (catalog no. 28601, Thermo Scientific) method.
Mice (C57BL6, 2–3 months old) were anesthetized as above, and a single bolus of 125I-WT-LRP-IV or 125I-LRPIV-D3674G in PBS was administered via femoral vein, and blood samples were collected from the retro-orbital sinus at different time points after injection within 24 h, as described earlier (14). Plasma samples were counted, and trichloroacetic acid (TCA)-precipitable radioactivity was determined. Pharmacokinetic parameters were determined using a non-compartmental intravenous bolus module of Kinetica version 5.1 software (Thermo Fisher Scientific).
125I-WT-LRPIV (20 μCi/kg) or 125I-LRPIV-D3674G (20 μCi/kg) was administered intravenously and compared with [14C]sucrose (11 μCi/kg) uptake by the brain and CSF 1 h after injection of tracers. 125I TCA-precipitable counts/min were determined in the plasma, CSF, and brain. In these studies, the brain was perfused briefly with cold PBS at the end of the experiment to remove residual vascular radioactivity, as confirmed by undetectable radioactivity of [14C]sucrose (11 μCi/kg). The BBB PS product (μl/min/g) of [14C]sucrose, 125I-WT-LRPIV, and 125I-LRPIV-D3674G was calculated by using the equation,
where VD is the volume of distribution of [14C]sucrose, 125I-WT-LRPIV, and 125I-LRPIV-D3674G after 1 h of intravenous injection in whole brain homogenate; CPL(T) is the terminal plasma concentration of TCA-precipitable 125I-WT-LRPIV, 125I-LRPIV-D3674G, or [14C]sucrose; and AUC is area under the curve on a plot of concentration versus time calculated as we described (43).
Ten-week-old C57BL6 mice (n = 5 mice/group) were treated daily with WT-LRPIV or LRPIV-D3674G (20 μg/mouse, intravenously via the tail vein) or vehicle for 5 days. At the end of treatment, mice were sacrificed under anesthesia. Blood and CSF samples were collected, and plasma was immediately separated from blood at 4 °C. All samples were stored immediately at −80 °C. Mice were perfused intracardially with ice-cold heparinized saline and hemibrains homogenized in 2% SDS containing protease inhibitor mixture (Roche Applied Science). Mouse endogenous Aβ40 and Aβ42 levels in the plasma and brain were determined by mouse Aβ-specific ELISA as described below. WT-LRPIV and LRPIV-D3674G levels in CSF were determined by Western blot analysis as described earlier (14).
Briefly, for mouse Aβ40-specific sandwich ELISA, the capturing and biotinylated detecting antibodies were monoclonal mouse Aβ raised against amino acid residues 1–20 (catalog no. AMB0062, Invitrogen) and rabbit polyclonal anti-Aβ40 biotin conjugate (catalog no. 44-3489, Invitrogen), respectively. For mouse Aβ42-specific sandwich ELISA, the capturing and detecting antibody were AMB0062 and rabbit polyclonal anti-Aβ42 biotin conjugate (catalog no. 44-3449, Invitrogen), respectively. Murine synthetic Aβ40 and Aβ42 (American Peptide Co., Sunnyvale, CA) were used as standards, as reported (44).
Total cholesterol levels in plasma were determined using a kit (catalog no. 439-17501, Wako Chemicals USA Inc., Richmond, VA).
Sandwich ELISA for murine plasma apoE was carried out as described (14). Plasma samples were diluted directly with BSAT-DPBS buffer (Dulbecco's phosphate-buffered saline (Cellgro, Mediatech, Inc.) with 5% BSA and 0.03% Tween 20, supplemented with complete protease inhibitor mixture (Roche Applied Science)). Pooled plasma from C57BL6 mice containing 68 μg/ml apoE was used as a standard as reported previously (14, 45). Briefly, 96-well plates (Costar, Corning) were coated overnight with 0.5 μg/well of the capturing antibody (catalog no. sc-6384, clone M-20, goat anti-mouse apoE, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), blocked with 7% milk in PBS, and incubated with the secondary antibody, biotinylated goat anti-apoE (1:10,000 dilution, catalog no. K74180B, Biodesign Meridian LifeScience), followed by streptavidin-horseradish peroxidase conjugate (catalog no. SNN 2004, Invitrogen). The reaction was developed using 3,3′,5,5′-tetramethylbenzidine substrate (KPL), stopped with 1 n HCl, and quantified at 450 nm. The sensitivity of the assay was ~1 ng/ml.
Plasma levels of active mouse tPA were determined using a kit (catalog no. MTPAKT, Molecular Innovations, Novi, MI).
Plasma levels of mouse pro-MMP-9 were determined using a kit (Quantinine® MMP900, R&D Systems) and following the manufacturer's instructions.
Plasma glucose levels were determined using GlucCellTM Test Trip (CESCO, Bioengineering Co., Taichung, Taiwan).
Mouse blood samples were collected in citrate buffer (250 mm, pH 7.2) and centrifuged at 10,000 × g for 10 min to separate plasma. Plasma (25 μl) was incubated with 25 μl of TriniCLOT aPTT HS reagent (Trinity Biotech plc., Bray, Ireland) for 3 min at 37 °C in a Start4 coagulometer (Diagnostica Stago, Inc.). The aPTT reaction was started by adding 50 μl of 50 mm calcium chloride, and the time of clotting was recorded when the movement of the iron ball ceased under the magnetic field.
Briefly, tissues were homogenized in PhosphosafeTM extraction buffer (catalog no. 71296, Novagen, EMD Millipore, Billerica, MA). Proteins were separated by SDS-PAGE and immunoblotted with an LRP1 light chain-specific antibody (catalog no. 438192, Calbiochem, EMD Millipore), anti-phosphotyrosine antibody (Sigma-Aldrich), low density lipoprotein receptor-specific antibody (catalog no. 15C8, Calbiochem, EMD Chemicals Inc.), and anti-RAGE antibody (catalog no. sc-5563, Santa Cruz Biotechnology, Inc.).
For detection of APP, frozen brain cortical tissues were homogenized in lysis buffer (Roche Applied Science) containing a complete protease inhibitor mixture (Roche Applied Science). The homogenate was centrifuged for 20 min at 20,000 × g. The supernatant was collected in a separate tube, and total protein concentration was determined by a BCA protein assay (Pierce). Brain lysate proteins were subjected to 4–12% NuPAGE BisTris SDS-PAGE (Invitrogen) and transferred to nitrocellulose membrane (Bio-Rad). Membrane was blocked with 5% nonfat milk in TBST for 1 h and incubated overnight with the primary monoclonal antibody against APP (MAB348, Chemicon, Temecula, CA). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Immunoreactivity was detected using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).
CSF samples (15 μl) were denatured in the SDS buffer by heating at 70 °C for 10 min. Samples were subjected to electrophoresis on an SDS-polyacrylamide gel and transferred to nitrocellulose. LRPIV was detected with a rabbit polyclonal anti-LRPIV antibody. In a separate experiment, 125I TCA-precipitable counts in 20 μl of plasma and CSF were analyzed 1 h after a single bolus of 125I-labeled WT-LRPIV or LRPIV-D3674G (20 μCi/kg) in PBS.
Immunogenicity testing was out-sourced (QED Bioscience Inc., San Diego, CA). Approximately 8–10-week-old BALB/c female mice (average weight 0.02 kg) were dosed (20, 40, and 60 μg/kg, intraperitoneally) four times biweekly. Retro-orbital blood samples of 250 μl each were collected before dosing and 1 week after following each injection. Each mouse was bled a total of five times. Blood samples were collected in heparinized tubes and centrifuged immediately after collection. Serum samples were frozen at −20 °C. After the last blood sample collection, all of the serum samples were tested for anti-LRPIV-D3674G antibody by ELISA. The plates were coated with three different LRPIV-D3674G concentrations (1, 5, or 20 μg/ml), and plasma samples were tested from 1:100 to 1:6400 dilutions for anti-LRPIV-D3674G antibodies. A rabbit LRPIV antiserum was used as a positive control.
APPsw+/0 mice (n = 9 mice/group) were treated subcutaneously with LRPIV-D3674G (40 μg/kg per day) or saline for 3 months beginning at the age of 6 months. After 3 months of treatment, we measured cerebral blood flow responses to brain activation by whisker stimulation and performed a burrowing behavioral test. After these functional tests, mice were sacrificed under anesthesia and brain, CSF, and plasma were collected and analyzed for human Aβ40 and Aβ42 levels as described earlier (14).
APPsw+/0 mice after 3 months of treatment with LRPIV-D3674G were subjected to a burrowing behavior test. The burrowing test was performed as described (46). Briefly, mice were individually placed in the cages equipped with a burrow made from a 200-mm-long, 68-mm-diameter tube of PVC plastic. One end of the tube was closed by a PVC cap. The open end of the tube was raised ~30 mm by a PVC ring. The burrow was filled with 200 g of mouse food pellets, and the mice were allowed to burrow for 3 h. The weight of the remaining food pellets inside the burrow was determined to obtain a measurement of the amount burrowed.
Formaldehyde fixed brain sections were used for immunostaining of the microglia marker Iba-1 (ionized calcium-binding adaptor molecule 1), as described (47).
Briefly, acetone-fixed saggital brain sections from vehicle-treated and LRPIV-D3674-treated mice were incubated in a 5% potassium ferrocyanide and 5% hydrochloric acid solution (1:1 working solution) for 30 min, as reported (48). Hemosiderin shows a blue color. To analyze the abundance of Prussian blue-positive deposits per section, the total numbers of Prussian blue-positive spots were divided by the number of sections analyzed.
The results were compared by one-way multifactorial analysis of variance followed by Tukey's post hoc analysis for more than two groups of data or Student's t test for unpaired data of two groups. The differences were considered to be significant at p < 0.05. All values are mean ± S.E.
We generated eight recombinant CR fragments of LRPIV with Myc and His6 tags (i.e. CR24–CR27, CR25–CR28, CR25–CR27, CR25–CR26, CR26-CR27, CR25, CR26, and CR21–CR31, corresponding to full-length LRPIV (Fig. 1, A and B). We also generated WT-LRPIV without Myc and His6 tags or the 16-amino acid peptide tag derived from factor VIII (tagged WT-LRPIV) that has been described previously (38, 49, 50). Two single CRs (CR25 and CR26), two double CRs (CR25-CR26 and CR26-CR27), and CR21–CR31 were eliminated from further screening because of their poor secretion by CHO cells into the medium. The remaining three LRPIV fragments were purified from CHO-secreted medium, including two four-CR fragments (CR24–CR27 and CR25–CR28) and one three-CR fragment (CR25–CR27), and, along with the full-length WT-LRPIV (Fig. 1C), were used for the binding studies. We set the criterion that a lead analog has to have a higher binding affinity for both Aβ40 and Aβ42 (i.e. lower Kd value) compared with previously described tagged WT-LRPIV, which has been shown to bind Aβ40 and Aβ42 in vitro with Kd values of 2 and 5 nm, respectively (14). The Kd for Aβ40 binding to CHO-secreted WT-LRPIV was 1.90 ± 0.40 nm (Table 2), which was comparable with Kd for Aβ40 binding to tagged WT-LRPIV produced by the baby hamster kidney cell line (14). However, Aβ40 bound with 3.5–7-fold lower affinity to CR24–CR27, CR25–CR28, and CR25–CR27 than to WT-LRPIV (Table 2) or tagged WT-LRPIV (14), which eliminated these CRs from further screening, although Aβ42 binding to these CRs was somewhat improved relative to WT-LRPIV (Table 2) and/or tagged WT-LRPIV (14).
Next, we performed site-directed mutagenesis, replacing the aspartic acid residue with glycine in the calcium binding sites of CR21, CR22, CR26, CR27, CR28, and CR29 and generated six full-length LRPIV mutants (i.e. D3354G, D3394G, D3556G, D3595G, D3633G, and D3674G, respectively) (Fig. 1B). Three LRPIV mutants (D3354G, D3585G, and D3633G) were eliminated from screening because of low levels of secreted protein. The remaining three LRPIV analogs, including the D3394G, D3556G, and D3674G LRPIV variants, were purified (Fig. 1C) and screened. LRPIV-D3674G mutant bound Aβ40 and Aβ42 with the highest affinity with a Kd of 1.20 ± 0.18 and 2.51 ± 0.6 nm, respectively, indicating 3–4-fold better affinity for the respective Aβ peptides than D3394G or D3556G mutants (Fig. 1, D and E, and Table 2). Importantly, compared with WT-LRPIV or tagged WT-LRPIV (14), LRPIV-D3674G bound Aβ40 and Aβ42 with ~1.6- and 2.7-fold greater affinity, respectively (Table 2) and therefore met our criterion for a lead Aβ-binding LRPIV analog.
To determine which region of Aβ is recognized by LRPIV-D3674G, we have studied in vitro binding of Aβ40 to immobilized LRPIV-D3674G with and without N terminus anti-Aβ40, C terminus anti-Aβ40, and control NI-IgG antibodies (Fig. 1F). This experiment has shown that LRPIV-D3674G binds to the C-terminal sequence of Aβ, which is consistent with a previous report showing that the wild type form of recombinant LRP-IV containing an extraneous 16-amino acid “tag,” which contains the antigenic determinant of the mouse monoclonal antibody CLB-CAg 69 against Factor VIII (35, 51), also binds the Aβ C-terminal domain (40). The precise amino acid sequence that LRPIV-D3674G detects will be determined by future epitope mapping studies. We also show that LRPIV-D3674G can bind, in addition to Aβ monomers, also Aβ oligomers and Aβ fibrils (Fig. 1G).
Because LRPIV binds multiple ligands besides Aβ, we studied binding of different LRP1 ligands for WT-LRPIV and LRPIV-D3674G, a lead new LRPIV analog, to determine whether LRPIV-D3674G mutation increases or decreases binding of other LRP1 ligands. Compared with WT-LRPIV, LRPIV-D3674G bound apoE2, apoE3, apoE4, tPA, MMP-9, and factor IXa, with ~1.3–4.1-fold lower affinity (Table 3). These data indicate that the LRPIV-D3674G mutation also improves Aβ binding relative to other studied LRP1 ligands.
There was no significant difference in the systemic clearance rate (t½ ~3.4 h) or mean residence time (~4.8 h) of 125I-WT-LRP-IV or 125I-LRPIV-D3674G after an intravenous injection of tracers (Fig. 2 and Table 4). However, the t½ was shorter compared with previously reported t½ for the wild type form of recombinant LRP-IV containing an extraneous 16-amino acid tag, which contains the antigenic determinant of the mouse monoclonal antibody CLB-CAg 69 against Factor VIII used to purify the peptide from the culture medium (14). It remains unclear how the Factor VIII tag prolongs the rate of systemic clearance of WT-LRP-IV (t½ = 11.8 h) (14), but it is of note that none of the presently studied forms of LRP-IV contained this extraneous 16-amino acid tag of Factor VIII.
Five-day intravenous treatment of C57Bl6 mice (20 μg/mouse) with WT-LRPIV or LRPIV-D3674G at a dose comparable with that used previously to treat C57Bl6 mice with tagged WT-LRPIV (14) significantly reduced mouse endogenous brain Aβ levels. For example, compared with vehicle, LRPIV-D3674G mutant lowered brain Aβ40 and Aβ42 levels by 42 and 45%, respectively (Fig. 3, A and B), which correlated with the corresponding increases in total plasma levels of Aβ40 and Aβ42 (Fig. 3, C and D), most of which was bound to the LRPIV-D3674G mutant (not shown), similar to what was reported previously for tagged WT-LRPIV in C57Bl6 mice treated with tagged WT-LRPIV (14). Compared with WT-LRPIV, LRPIV-D3674G was more efficacious in removing mouse endogenous brain Aβ, as indicated by ~25 and 27% greater reductions in brain Aβ40 and Aβ42 levels compared with the respective Aβ peptide reductions with WT-LRPIV (Fig. 2, A and B).
We analyzed plasma and tissue samples from C57Bl6 mice treated intravenously with 20 μg of LRPIV-D3674G/mouse daily for potential side effects. LRPIV-D3674G did not alter plasma levels of cholesterol, glucose, or LRP1 ligands, such as apoE, tPA, and pro-MMP-9 (Fig. 4, A–E). Plasma clotting time determined by the aPTT remained unchanged within 2 h of intravenous LRPIV-D3674G administration (Fig. 4F). In liver and brain microvessels, there were no changes in the expression levels of low density lipoprotein receptor or LRP1 (Fig. 5, A and B). In addition, in the brain, there were no changes in phosphorylated LRP1 levels after LRPIV-D3674G treatment (Fig. 5C). Expression of RAGE, an Aβ influx transporter (4), was not altered in brain microvessels by LRPIV-D3674G treatment (Fig. 5D). Furthermore, APP levels in the brain were not altered by LRPIV-D3674G treatment (Fig. 5E). LRPIV-D3674G did not enter CSF (Fig. 5F), similar to tagged WT-LRPIV (14), suggesting that its action is on sequestering the peripheral Aβ pool. Using radiolabeled 125I-WT-LRPIV and 125I-LRPIV-D3674G, we have independently confirmed that neither peptide is transported into the CSF and brain, as determined by non-detectable radioactivity in the CSF and non-detectable PS products for either form of LRP-IV (Fig. 5, G and H). For immunogenicity testing, blood samples were collected at the time of antigen administration and 1, 3, 5, and 7 weeks afterward. Antibodies to LRPIV-D3674G were not detected in postimmunization plasma samples in mice after treatment with increasing doses of LRPIV-D3674G, as described under “Experimental Procedures” (Fig. 5I).
Compared with vehicle-treated APPsw+/0 mice, APPsw+/0 mice treated daily with a subcutaneous low dose of LRPIV-D3674G (40 μg/kg) for 3 months beginning at 6 months of age showed significant reductions of Aβ40 and Aβ42 levels in hippocampus, cortex, and CSF by 60–80% (Fig. 6, A–D), as determined at 9 months of age. As expected, LRPIV-D3674G treatment significantly increased plasma Aβ40 and Aβ42 levels (Fig. 6, E and F). Treatment with LRPIV-D3674G also increased cerebral blood flow (CBF) responses to whisker stimulation by 75% (Fig. 6G) and significantly improved hippocampal function compared with vehicle-treated mice, as indicated by the burrowing test showing an approximately 60% improvement (Fig. 6H). The LRPIV-D3674 treatment in APPsw+/0 mice did not influence inflammatory response and did not increase microhemorrhages, as shown by non-significant differences in the number of Iba1-positive microglia (Fig. 7, A and B) and Prussian blue-positive hemosiderin deposits (Fig. 7, C and D) in LRPIV-D3674-treated compared with vehicle-treated animals.
Plasma sLRP1 levels are reduced in AD and MCI-AD patients, and sLRP1 is oxidized, which results in diminished Aβ peripheral binding and higher levels of free Aβ in plasma (14, 22). In addition, WT-LRPIV binds free Aβ in plasma of AD patients (14). Experimental studies have shown that free Aβ re-enters the brain (4, 23, 24, 52) and that sLRP1 and/or its recombinant LRPIV ligand binding domain prevent plasma Aβ from entering the brain (14). These studies underscored the need for development of sLRP1 replacement therapy for AD to maintain low levels of free, unbound Aβ in plasma and restore an important homeostatic mechanism regulating brain Aβ (11). Circulating endogenous Aβ antibodies that bind a fraction of Aβ in plasma (14, 22, 53–56) are also decreased in early stages of AD (22, 57, 58), further emphasizing the need for peripheral Aβ binding treatments. Peripheral Aβ-binding agents, such as Aβ antibodies (25, 48, 59–61), gelsolin (62, 63), soluble RAGE (4), and LRPIV (14), have been shown to promote Aβ clearance from the brain, reducing brain Aβ and amyloid load in different APP-overexpressing mice.
LRP1 at the BBB, sLRP1 in plasma, and LRP1 in the liver have important roles in homeostasis of brain Aβ (5). Down-regulation of LRP1 at the BBB due to vascular risk factors and/or oxidative stress (17, 64), oxidation, or reduced levels of sLRP1 in plasma (14) and reduced expression of LRP1 in the liver (65) lead to accumulation of Aβ in brain in different experimental models. A novel recombinant LRPIV-D3674G variant described here preferentially binds Aβ compared with other ligands and effectively reduces Aβ brain levels in wild type mice and AD mice without noticeable side effects. Moreover, treatment with LRPIV-D3674G increased CBF responses to whisker stimulation in AD mice. Whether the improved CBF responses contribute to reductions in Aβ levels in the brain by promoting Aβ clearance into cerebral circulation remains to be determined by future studies designed to measure Aβ clearance and production under experimentally different CBF conditions. LRPIV-D3674G did not enter CSF or brain, which is not unexpected because WT-LRPIV also does not cross the BBB (14), and peptides and proteins in general need specific transport systems expressed in brain endothelium for their transport into the brain (66).
The data presented here represent the first screening of LRPIV-derived analogs for Aβ binding. LRPIV has been shown to bind a range of functionally distinct ligands (35, 37, 38, 49). The surface plasmon resonance analysis has shown that the CR24–CR28 LRPIV region most effectively binds RAP, α2M*, Factor VIII light chain, and Factor IXa (35). The three triple repeats, CR24–CR26, CR25–CR27, and CR26–CR28, interact avidly with RAP compared with the other CRs of LRPIV (33). Although the Aβ binding site on LRPIV has not been characterized, it seems plausible that this site might overlap partially or fully with the RAP binding site(s) because we have previously shown that RAP abolishes Aβ binding to LRPIV (8). Although Aβ40 and Aβ42 interacted with all studied CR fractions (CR24–CR27, CR25–CR28, and CR25–CR27), their binding affinities varied. In contrast to Aβ42, full-length WT-LRPIV was required for high affinity binding of Aβ40. Other studies have reported that CR24–CR26 is involved in high affinity binding of α2M* and Factor VIII light chain (35). Site-directed mutation at a calcium binding site within CR29 (D3674G) increased the affinity for Aβ (Aβ42 > Aβ40) but decreased the affinity for other studied LRP1 ligands, whereas mutations within CR22 (D3394G) and CR26 (D3556G) reduced their respective binding affinities for both Aβ40 and Aβ42.
Calcium is required for proper folding and structural stability of the CR regions (33). The calcium ion binding requires the side chain of four acidic residues and two carbonyl groups, usually an aspartate residue and an aromatic residue. These two residues are involved in ligand interaction with LRP1 (67, 68). CRs (CR23, CR30, and CR31) with imperfect calcium coordination have reduced ligand-binding affinities (35). Also, calcium is required for binding of some LRP1 ligands, such as RAP and α2M* (37), but not for other LRP1 ligands, such as apoE and lactoferrin (37). Importantly, mutation in CR29 resulted in conformational change in LRPIV, which enhanced binding of Aβ, especially Aβ42, but reduced binding of other ligands, including apoE2, apoE3, apoE4, tPA, MMP-9, and Factor IXa. The nature of the conformational change in the LRPIV-D3674G variant and mechanism of the enhanced Aβ binding will require future structural and biophysical studies.
In summary, we have demonstrated that LRPIV-D3674G exhibits significantly improved Aβ binding compared with WT-LRPIV. Moreover, LRPIV-D3674G efficiently cleared Aβ from the brain in wild type and AD mice. In addition, our data show that LRPIV-D3674G binds with reduced affinity several studied LRP1 ligands in vitro and does not affect noticeably their activity in vivo in mice. Therefore, LRPIV-D3674G could be developed as a potential therapeutic agent administered as a monotherapy and/or in combination with other Aβ-lowering agents, such as Notch-sparing γ-secretase inhibitors or β-secretase (BACE1) inhibitors (57, 69, 70) to restore the natural peripheral sink mechanism for Aβ in MCI and AD patients (22, 71).
We thank Dr. R. Deane for helpful discussions and M. Watrobski for skillful technical assistance.
*This work was supported, in whole or in part, by National Institutes of Health Grants AG023084 and NS034467. This work was also supported by ZZ Alztech (Rochester, NY). Berislav V. Zlokovic is the scientific founder of Socratech LLC, a startup biotechnology company with a mission to develop new therapeutic approaches for stroke and Alzheimer disease. Berislav V. Zlokovic is co-inventor on patents pertaining to use of sLRP fragments as a potential therapy for Alzheimer disease.
3The abbreviations used are: