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NEUROPHARMACOLOGY

In Vivo β-Secretase 1 Inhibition Leads to Brain Aβ Lowering and Increased -Secretase Processing of Amyloid Precursor Protein without Effect on Neuregulin-1

Departments of Alzheimer's Research (S.Sa., E.A.P., G.W., M.-C.C., X.-P.S., K.T., K.X.T., J.K., A.J.S.), Drug Metabolism (J.E., L.J.), and Medicinal Chemistry (T.S., S.St., C.C.), Merck Research Laboratories, West Point, Pennsylvania

Received August 13, 2007; accepted December 20, 2007.

	Abstract

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β-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid β peptide Aβ42, the putative neurotoxic species in Alzheimer's disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC₅₀ 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose- and time-dependent lowering of brain Aβ40 and Aβ42. BACE1 inhibition also led to a robust brain secreted (s)APPβ lowering that was accompanied by an increase in brain sAPP levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (–/–) mice, in agreement with previous studies, this effect was also observed in 15-day-old heterozygous (+/–) mice, but it was not evident in 30-day-old and 2-year-old BACE1 KO (–/–) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant Aβ lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major β-site cleavage enzyme for APP and that its inhibition can lower brain Aβ and redirect APP processing via the potentially nonamyloidogenic -secretase pathway, without significantly altering NRG-1 processing.

BACE1-deficient mice generated from multiple groups were viable, and they showed subtle alterations in behavioral and neurochemical phenotype (Roberds et al., 2001; Harrison et al., 2003), but robust reduction in neuronal Aβ production (Cai et al., 2001; Luo et al., 2001) and amyloid plaque deposition (Ohno et al., 2004; Laird et al., 2005). In contrast, mice overexpressing human BACE1 showed an increase in brain sAPPβ, CTFβ, and Aβ levels, suggesting enhanced amyloidogenic processing of APP and amyloid plaque deposition (Bodendorf et al., 2002; Mohajeri et al., 2004; Willem et al., 2004), whereas reduced plaque deposition has been observed in an independent mice line (Lee et al., 2005). In contrast, in mice overexpressing the -secretase enzyme ADAM10, a reduction in Aβ peptides and amyloid plaque deposition was observed previously (Postina et al., 2004). Thus, either inhibition of BACE1 enzymatic activity or an elevation of -secretase activity can reduce brain Aβ production (Hardy and Selkoe, 2002).

Although there is evidence for early lethality (Dominguez et al., 2005) and age-dependent development of cognitive deficits (Laird et al., 2005) in BACE1 KO mice, the overall benign phenotype from multiple studies had supported the idea that BACE1 inhibition is a safe therapeutic target for Aβ lowering in human AD (Citron, 2004). However, recent studies have shown that BACE1 KO mice show hypomyelination and putative alteration in neuregulin (NRG)-1) processing, as evidenced by an increase in brain full-length NRG-1 levels (Willem et al., 2006). These deficits in NRG-1 processing are thought to lead to defects in both peripheral (Willem et al., 2006) and central nervous system myelination in young mice (Hu et al., 2006). Thus, a mechanism-based effect on peripheral or central myelination could potentially affect BACE1 inhibitor development as an AD therapeutic. Although BACE1 inhibitors have shown acute in vivo efficacy to lower brain Aβ levels in murine models when administered peripherally (Chang et al., 2004; Stachel et al., 2006; Hussain et al., 2007) or via direct intracranial injection (Asai et al., 2006; Nishitomi et al., 2006), their poor pharmacokinetic properties have prevented effective evaluation of subchronic to chronic BACE1 inhibition on brain APP processing and NRG-1 processing in adult mice.

In a recent study, we have shown that small-molecule hydroxyethylamine dipeptide isosteres are very potent inhibitors of BACE1 (IC₅₀ 15 nM) (Stachel et al., 2004; Pietrak et al., 2005; Shi et al., 2005). In the present study, we demonstrate that direct infusion of this inhibitor, over 1 to 2 weeks, into the lateral ventricles of brains of mice led to a sustained reduction of brain Aβ40, Aβ42, and sAPPβ, along with a corresponding elevation of sAPP. Thus, the impact of subchronic to chronic BACE1 inhibition in mice on both APP processing and NRG-1 processing could be evaluated. The questions that are addressed in this study include whether there is a gene-dosage effect on NRG-1 processing in young BACE1 KO mice; whether defects in NRG-1 processing are observed in aged BACE1 KO mice similar to that in young mice; and finally, whether in vivo BACE1 inhibition in adult mice leads to deficits in NRG-1 processing. We confirmed previous findings that there is an increase in full-length NRG-1 protein levels in young postnatal day P15 BACE1 KO (–/–) mice compared with WT mice (Willem et al., 2006). In P15 BACE1 (+/–) mice, we observed an increase in full-length NRG-1 protein similar to (–/–) mice, suggesting a lack of BACE1 gene dosage on NRG-1 processing. In contrast, P30 and 2-year-old BACE1 KO mice showed no significant differences in either the full-length or N-terminal fragment of NRG-1 among the genotypes. These results indicate that NRG-1 processing may decline rapidly with age in mice, and they suggest an early developmental alteration in NRG-1 processing in BACE1 KO mice. Finally, we demonstrate that BACE1 inhibition over 1 week was not accompanied by any alterations in NRG-1 levels in adult mice. These data support the hypothesis that inhibition of BACE1 can potentially ameliorate the amyloid burden in the brain of AD patients by both decreasing Aβ production and shifting APP processing to the -secretase pathway, while not affecting NRG-1 processing.

	Materials and Methods

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Antibodies. Aβ N-terminal antibody 6E10 (Kim et al., 1988) (catalog number SIG-39320; Covance Research Products, Princeton, NJ), which recognizes Aβ amino acids 3 to 8, was used as a capture antibody for Aβ40, Aβ42, and sAPP sandwich ELISAs. Neoepitope-specific antibodies were used to detect Aβ40 and Aβ42 peptides, similar to that described previously (Ida et al., 1996). We raised rabbit polyclonal antibodies to peptides encoding the sAPPβ_KM neoepitope by using an artificial norleucine amino acid in place of methionine. This antibody was affinity purified, it showed neoepitope specificity compared with the linear epitope around the BACE cleavage site, and it was used as a capture antibody for sAPPβ ELISAs. The 22C11 antibody to amino acids 66 to 81 of APP N terminus (MAB348; Millipore Bioscience Research Reagents, Temecula, CA) was used to detect both sAPP and sAPPβ in ELISA assays. Detection antibodies for ELISA assays were conjugated with alkaline phosphatase (AP) using the EZ-link maleimide-activated AP kit (31493; Pierce Chemical, Rockford, IL) using standard protocols.

Mice Models. The APP-YAC mice expressing the human WTAPP transgene under control of endogenous human APP promoter elements, in a yeast artificial chromosome vector, was used in these studies (Lamb et al., 1993). These mice show APP expression and Aβ levels that are 2 to 3-fold above endogenous murine levels, and they display no amyloid plaques with age (Lamb et al., 1993) compared with other mutant-APP transgenic mice driven by strong promoter elements (Kawarabayashi et al., 2001). All in vivo inhibitor studies were performed on 3- to 6-month-old mice. The APP-YAC mice showed no age-dependent changes in soluble brain Aβ levels, and they allowed efficient measurement of the human Aβ peptide from brain homogenates. Mice lacking APP (Zheng et al., 1995) were used to generate brain homogenates for running standard curves in ELISA assays. The BACE1 knockout mice (Cai et al., 2001) were obtained from Johns Hopkins University (Baltimore, MD), and they were backcrossed to the C57BL/6 background.

Intracerebroventricular Administration of BACE Inhibitor in Mice. Intracerebroventricular infusions of compound were done using osmotic minipumps (1002, 1003D, 1007D; Alzet, Cupertino, CA) with capacity of 100 µl and pumping rates of 0.25 to 1 µl/h. BACE inhibitor Merck-3 (Stachel et al., 2004) was dissolved in 50% polyethylene glycol 300 and 50% dimethyl sulfoxide (D2650; Sigma-Aldrich, St. Louis, MO) at concentrations of 12.5 to 50 mg/ml to deliver doses ranging from 7.5 to 30 mg/kg/day (mkd) of compound. Osmotic minipumps were filled with compound and connected via polyethylene tubing to customized infusion cannulas (2.2 mm; Plastics One, Roanoke, VA), and they were primed overnight at 37°C. The next day, droplets were observed at the outlet of the cannula, indicating that the pumps were primed and exuding compound. APP-YAC mice were surgically prepared, and then they were placed in a mouse stereotaxic station (model 900; David Kopf Instruments, Tujunga, CA). Mice were anesthetized via a mouse mask using 2 to 5% isoflurane. A midline incision was made in the scalp to expose the skull. The bregma was identified and coordinates AP, 1.2 mm; ML, –0.5 mm; and DV, –2.2 mm were used to target the left lateral ventricle (Paxinos and Franklin, 2001). A trephine drill was used to remove 1 mm in diameter of bone centered on the marked coordinates to expose the brain. The osmotic minipump was placed within an s.c. pocket under the skin on the back of the mouse. The attached infusion cannula was stabilized in a holder, and then it was gently inserted into the brain until it was flush with the skull and stabilized with dental cement. The tubing was buried under the skin, taking care not to introduce kinks, and the overlying skin was sealed with cyanoacrylate cement (Vetbond tissue adhesive; Henry Schein, Melville, NY). The mice were removed from the stereotaxic apparatus, and they were allowed to recover over warm water circulating blankets. Then, they were returned to their home cage until the end of the study. All animal procedures were done using sterile technique and procedures were fully approved by the Institutional Animal Care and Use Committee at Merck.

After the desired time of treatment with compounds, the mice were euthanized using a CO₂ chamber. Blood was removed by cardiac puncture, collected in K₂EDTA tubes, and centrifuged at 5000 rpm for 10 min. Plasma was stored frozen at –80°C. Brains were removed and sectioned mid-sagittally. Each hemisphere was weighed and stored at –80°C in polypropylene tubes until further analysis.

Measurement of Brain Aβ40, Aβ42, sAPPβ, and sAPP by Sandwich ELISA. Mouse brains were extracted in 0.2% diethylamine in 10x volume (w/v), heat-treated, and spun at 170,000g for 90 min. Supernatant was collected and neutralized with 10% volume 0.5 M Tris-HCl, pH 6.8, and then it was either run in Western blots or plated for ELISA for specific analytes. Brain Aβ40, Aβ42, and sAPP analytes were captured in ELISA plates with the 6E10 antibody, whereas the KM neoepitope antibody was the capture antibody for sAPPβ. Black polystyrene Costar plates (Costar 3925; Corning Life Sciences, Acton, MA) were coated overnight with the 5 µg/ml capture antibody, washed, and then blocked with 3% bovine serum albumin in PBS. The plates were stored at 4°C until use. One hundred microliters of sample was added per well in duplicate, followed by 50 µl of detection antibody conjugated with alkaline phosphatase (final IgG concentration at 0.1 µg/ml). Brain Aβ40 and Aβ42 were detected with C-terminal neoepitope antibodies, whereas N-terminal linear epitope antibody 22C11 was used to detect both sAPPβ and sAPP. Aβ40 and Aβ42 standards (American Peptide Co., Inc., Berkeley, CA) and sAPPβ and sAPP peptide standards (S4316 and S9564; Sigma-Aldrich) were stored in frozen aliquots at –80°C, and they were used in standard curve dilutions in APP-KO brain matrix, prepared in an identical manner as the study samples. After overnight incubation at 4°C, plates were washed and developed using alkaline phosphatase substrate (T2214; Applied Biosystems, Foster City, CA). Luminescence counts were measured using LJL Analyst (Molecular Devices, Sunnyvale, CA).

Standard curves were fit using a third order spline fit, coefficients were determined, and unknown sample counts were converted to actual analyte concentrations. All statistical analysis was performed on log-transformed data. The brain Aβ40 and Aβ42 assays had a lower limit of sensitivity of 3 and 1.56 pM (33 and 17 fmol/g after adjusting for brain weight), respectively. Brain levels of Aβ40 were in the range of 750 to 1500 fmol/g, whereas brain Aβ42 was in the range of 100 to 400 fmol/g. Brain sAPPβ and sAPP assays had a lower limit of sensitivity of 12.5 pM (300 fmol/g adjusting for brain weight). Brain sAPPβ and sAPP were in the range of 100 to 8000 fmol/g. Because the standards showed lot-to-lot variability in signal and calculated amounts, these data were normalized to the vehicle group for analysis.

Western Blotting for Brain sAPPβ and sAPP. For Western blots, 25 µl of mouse brain homogenate, as described above under Measurement of Brain Aβ40, Aβ42, sAPPβ, and sAPP by Sandwich ELISA, was mixed with 25 µl of 2x sample loading buffer with reducing agent, it was boiled for 5 min, and then it was resolved using 10 to 20% Tris-Tricine SDS-polyacrylamide gel electrophoresis gels (Bio-Rad, Hercules, CA). Separated proteins were electrophoretically transferred onto 0.2-µm nitrocellulose membranes, backed by 0.1-µm nitrocellulose. The membranes were boiled for 5 min in PBS, and then they were blocked with Odyssey blocker (LI-COR, Lincoln, NE), followed by overnight incubation with primary antibodies 6E10 for sAPP and rabbit KM-neoepitope antibody for sAPPβ. Secondary antibodies IRDye goat anti-mouse 800 nm (Lonza Rockland, Inc., Rockland, MD) and Alexa Fluor goat anti-rabbit 680 nm (A21076 [GenBank] ; Invitrogen, Carlsbad, CA) were diluted 1:2500 in Odyssey blocker with 0.1% Tween 20 for 1 h and washed with PBS containing 0.1% Tween 20. Blots were then visualized and quantified using an Odyssey infrared scanner (LI-COR).

Western Blotting for Brain BACE1, Neuregulin, and Myelin Basic Protein. To quantify BACE1 and neuregulin, brain hemispheres were homogenized using a modified radioimmunoprecipitation assay buffer (1% Nonidet-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM Na₃VO₄, 1 mM NaF in 50 mM Tris-HCl, pH 7.4). Ten microliters of brain homogenates was mixed with 10 µl of 2 x sample loading buffer, boiled for 5 min, and resolved using 4 to 20% gradient Tris-glycine SDS-polyacrylamide gel electrophoresis gel (Invitrogen). Separated proteins were electrophoretically transferred onto 0.2-µm polyvinylidene difluoride membranes. The membranes were blocked with Odyssey blocker (LI-COR), followed by overnight incubation with primary antibodies. Blots were incubated with rabbit polyclonal antibody to BACE1 (EE17; Sigma-Aldrich) with or without mouse monoclonal antibody to myelin basic protein (MBP) (smi94; Covance Research Products). A second set of blots was incubated with rabbit polyclonal antibody to the N terminus of NRG-1 (sc28916; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Tubulin staining with mouse monoclonal antibody to βIII-tubulin (MMS-435P; Covance Research Products) was used as a loading control. Secondary antibodies IRDye goat anti-mouse 800 nm (610-132-121; Lonza Rockland, Inc.) and Alexa Fluor goat anti-rabbit 680 nm (catalog number A21109; Invitrogen) were diluted 1:2500 in Odyssey blocker with 0.1% Tween 20 for 1 h and washed with PBS containing 0.1% Tween 20. Blots were then visualized and quantified using an Odyssey infrared scanner (LI-COR).

Data Presentation and Statistical Analysis. Statistical analysis was performed using JMP version 5.0 (SAS Institute, Cary, NC). Graphs and figures were prepared using JMP or Origin 7.5 (OriginLab Corp., Northampton, MA) and collated using Adobe Illustrator (Adobe Systems, Mountain View, CA).

Box and whisker plots were used to display data from individual animal data using JMP. The box plots in red summarize the data from each treatment group. The ends of the box are the 25th and 75th quantiles. The red line across the middle of the box indicates the median. Each box has red lines or whiskers that extend from the ends of the box to the highest and lowest magnitude data point. The horizontal green line represents the mean of the data for each group.

Average data were plotted as the mean ± S.E.M. Statistical significance was denoted by *, p < 0.05, **, p < 0.01, and ***, p < 0.001 using Tukey-Kramer honestly significant difference (HSD) or Student's t test.

	Results

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Intracerebroventricular Administration of BACE1 Inhibitor Merck-3 Led to Robust Brain Aβ40 and Aβ42 Lowering in Mice. We have reported previously that Merck-3 is a potent inhibitor of in vitro BACE1 enzyme activity, with an IC₅₀ of 10 nM and reduced sAPPβ production from cells, with an IC₅₀ of 30 nM. Merck-3 was selective for BACE1, and it had an in vitro IC₅₀ value of 230 nM for BACE2, 7 µM for cathepsin D, and >50 µM for renin, respectively (Stachel et al., 2004; Pietrak et al., 2005). Merck-3 showed a robust inhibition of secreted sAPPβ, and it displayed a dose-dependent reduction of secreted Aβ40 in primary brain slice cultures, with an IC₅₀ of 20 nM (Supplemental Fig. 1). These results suggested that Merck-3 can inhibit endogenous BACE1 in mice brain slice cultures.

View larger version (21K): [in this window] [in a new window]   Fig. 1. The i.c.v. administration of BACE1 inhibitor Merck-3 led to robust brain Aβ40 and 42 lowering. Brain Aβ40 and 42 levels were measured 24 h after i.c.v. infusion, into the left lateral ventricles, of Merck-3 at 30 mkd into n = 6 mice compared with n = 6 vehicle-treated mice. A, brain Aβ40 (left hemisphere) was lowered 64% with Merck-3 at 30 mkd (p < 0.001; Student's t test). B, brain Aβ42 (left hemisphere) was lowered 55% with Merck-3 at 30 mkd (p < 0.001; Student's t test). C, there was a significant correlation between brain Aβ40 and Aβ42 levels in the left brain hemisphere (r = 0.91; n = 12; p < 0.001; ANOVA). D, there was a significant correlation between the left and right brain hemispheres for both brain Aβ40 (black filled squares: r = 0.97, n = 12, p < 0.001; ANOVA) and brain Aβ42 (red open squares: r = 0.89, n = 12, p < 0.001; ANOVA). The box and whisker plots are further described under Materials and Methods.

We next tested whether brain Aβ can be lowered if Merck-3 was administered directly into the lateral ventricles of the brains of APP-YAC mice. We surgically implanted a cannula to deliver Merck-3 into the left lateral ventricle, by minipump infusion. Subcutaneous miniosmotic pumps (100 µl; 1 µl/h rate) were filled with Merck-3 at a concentration of 50 mg/ml, and they were connected via polyethylene tubing to a cannula and primed overnight at 37°C. The pump delivered vehicle or Merck-3 at a dose of 30 mkd into the left lateral ventricles. After recovery, the mice were sent to their home cage; they were sacrificed 24 h later, and brains were harvested. The mice tolerated the i.c.v. infusions well, and they showed normal feeding and exploratory behavior after recovery from anesthesia. Overall, survival was in the order of 95% among all surgically implanted animals, and there was no statistical difference between vehicle-treated and Merck-3-treated animals (vehicle, 53 of 55 and Merck-3, 68 of 71 animals survived from n = 11 experiments; p = 0.7). Brain concentrations of Merck-3 were in the range of 50 to 300 µM in this study. This is probably due to the large dose of compound infused into the brain and the possibility of localized precipitation in the ventricle leading to very high brain exposures of Merck-3.

Brain Aβ measurements were done in both the left and right hemispheres to determine whether there were spatial differences in BACE inhibition in the brain after delivery of Merck-3 into the left ventricle. Brain Aβ40 and Aβ42 in the left brain hemisphere was lowered 64 and 55%, respectively, with Merck-3 compared with vehicle-treated animals (p < 0.001 via Student's t test) (Fig. 1, A and B). Brain Aβ42 levels are generally in the range of 10 to 40% of brain Aβ40 levels in the APP-YAC mice (33 ± 2% in this experiment). Brain Aβ40 and 42 reductions of 50 to 60% were observed in the right cerebral hemispheres from the same animals with Merck-3, and no statistical differences were observed between brain Aβ40 and Aβ42 levels in the left versus right hemispheres, respectively (Aβ40, p = 0.2; Aβ42, p = 0.6).

There was a strong correlation between brain Aβ40 and brain Aβ42 levels in the left brain hemisphere (r = 0.91; n = 12; p < 0.001) (Fig. 1C). There was also good correlation between brain Aβ40 and Aβ42 measured in the left versus right hemisphere, respectively (Aβ40: slope = 1.04, r = 0.97, p < 0.001, n = 12; Aβ42: slope = 0.86, r = 0.89, p < 0.001, n = 16) (Fig. 1D). These data suggest that compound infused into the left lateral ventricle was able to diffuse efficiently into both hemispheres and lowered brain Aβ40 and 42. These results lead us to conclude that brain Aβ40 and 42 can be effectively lowered with a small-molecule BACE inhibitor if it can be delivered past the blood-brain barrier.

Time-Dependent Lowering of Brain Aβ and sAPPβ after Merck-3 i.c.v. Infusion. We next determined the time course of brain Aβ lowering after i.c.v. administration of Merck-3. Previous work has suggested that Aβ is rapidly turned over (Cirrito et al., 2003), and pharmacological studies have revealed Aβ lowering within a few hours after compound administration (Best et al., 2005). APP-YAC mice were infused with Merck-3, and brain Aβ was assessed at time points ranging from a few hours up to 2 weeks. The time course studies were done in two parts due to the large number of animals that had to be surgically prepared. In part 1, time points ranging from 2 h to 2 days were tested with Merck-3 infusion at a dose of 30 mkd (Fig. 2, filled symbols). In part 2 of the study, 1-, 7-, and 14-day infusion was tested at dose of 7.5 mkd Merck-3 (Fig. 2, open symbols), due to better tolerability at the lower dose at times greater than 1 day. The 30 mkd dose required the use of 1 µl/h pumps, and it was associated with lethargy and reduced activity in some animals at time points greater than 1 day. A similar lethargy was also observed in animals treated with vehicle using the 1-µl/h pumps, suggesting that it was not compound related. Infusions at the lower dose of 7.5 mkd, using the 0.25-µl/h pumps, were well tolerated over the durations of 1 to 2 weeks. Brain concentrations of Merck-3 were in the range of 50 to 200 µM in this study. The large range of values and the small n values at each time point did not allow robust pharmacokinetic-pharmacodynamic relationships to be determined from this study.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Time course of brain Aβ and sAPPβ lowering after i.c.v. administration of BACE1 inhibitor Merck-3. A, brain Aβ40 levels are plotted as a function of time after i.c.v. infusion. Composite graph of n = 2 separate studies, one study with Merck-3 at 30 mkd for 2 h, 6 h, 1 day, and 2 days, respectively (n = 3–5 mice/group; filled symbols) and a second study at 7.5 mkd at 1, 7, and 14 days, respectively (n = 3–5 mice/group; open symbols). Brain Aβ40 lowering, relative to the vehicle group, was 15% at 6 h, 55% at 1 day (p < 0.001), 64% at 2 days (p < 0.001), 60% at 7 days (p < 0.001), and 54% at 14 days (p < 0.001), respectively, after onset of infusion with Merck-3 (all via Tukey-Kramer HSD). B, brain Aβ42 levels as a function of time after i.c.v. infusion with vehicle or Merck-3 in the same experiment as described in A. Brain Aβ42 lowering, relative to the vehicle group, was 29% at 6 h, 63% at 1 day (p < 0.01), 85% at 2 days (p < 0.001), 72% at 7 days (p < 0.01), and 67% at 14 days (p < 0.01), respectively, after onset of infusion with Merck-3 (all via Tukey-Kramer HSD). C, Western blots for sAPPβ of brain homogenates from the above-mentioned experiment using a C-terminal neoepitope antibody. There was slight reduction in sAPPβ at 6 h and a robust reduction in sAPPβ levels at 1 and 2 days after infusion with 30 mkd Merck-3, consistent with the brain Aβ ELISA data in A and B. Untx, untreated.

A dose- and time-dependent reduction of brain Aβ40 and 42 was observed after i.c.v. infusion with Merck-3. Although no significant change in brain Aβ40 and 42 levels were observed at 2 h (dose achieved 2.5 mg/kg), there was significant reduction (20–30%) in both analytes at 6 h (dose achieved 7.5 mg/kg), relative to untreated or vehicle-treated animals (Fig. 2, A and B). At time points ranging from 1 to 14 days of infusion with Merck-3, a robust lowering of brain Aβ40 and 42 in the range of 50 to 70% was observed, relative to untreated or vehicle-treated animals (**p < 0.01 and ***p < 0.001 via Tukey-Kramer HSD) (Fig. 2, A and B). Brain Aβ40 and 42 levels were highly correlated in this study (r = 0.98; n = 37; p < 0.001). The lack of brain Aβ lowering at 2 h after infusion is probably due to the time taken for Merck-3 to diffuse from the ventricles into the brain parenchyma. Infusion with vehicle for 2 weeks produced no changes in brain Aβ40 or Aβ42 compared with untreated animals, suggesting that there was no effect of the i.c.v. infusion procedures or the vehicle used. These results suggest that a sustained lowering of brain Aβ analytes could be achieved after direct i.c.v. infusion of Merck-3.

The production of Aβ40 and Aβ42 requires the sequential cleavage of APP by BACE followed by -secretase. To directly test whether Merck-3 inhibits BACE, the production of the N-terminal cleavage product of APP, i.e., sAPPβ was examined in brain homogenates. Western blots of brain homogenates revealed that Merck-3 led to modest lowering at 6 h and a robust lowering of brain sAPPβ at the 1- and 2-day time points after i.c.v. infusion (Fig. 2C), similar to that observed in brain slice culture (Supplemental Fig. 1). Thus, brain Aβ lowering was correlated with brain sAPPβ lowering at corresponding time points. Taken together, these results are consistent with a direct effect of Merck-3 on BACE enzymatic activity in the brain and consequent reduction of brain sAPPβ and Aβ levels.

Brain sAPPβ Lowering Is Associated with Brain sAPP Elevation after Merck-3 Treatment. Previous studies in cell culture systems with pharmacological agents such as protein kinase C activators had suggested that BACE and -secretase enzymes compete with each other for APP processing (but see LeBlanc et al., 1998; Skovronsky et al., 2000; Zhu et al., 2001). Studies in BACE1 KO mice (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001) and mice overexpressing ADAM10, a putative -secretase enzyme, have demonstrated reductions in Aβ metabolites accompanied by elevation in -secretase cleavage products of APP and a reduction in brain amyloid pathology (Postina et al., 2004). Taken together, these data suggest that inhibition of BACE can lead to enhanced -secretase processing of APP and decreased production of amyloidogenic peptides. Because we observed a robust lowering of brain sAPPβ and Aβ with Merck-3, we examined whether direct inhibition of BACE with Merck-3 also alters -secretase processing of APP.

Brain sAPPβ and sAPP were evaluated in brain homogenates from mice treated with 7.5 mkd Merck-3 for 1, 7, and 14 days via i.c.v. infusion. Western blotting for brain sAPPβ, using a C-terminal neoepitope antibody, revealed a decrease in intensity after Merck-3 treatment (Fig. 3A). In contrast, sAPP detected using the 6E10 antibody showed a modest increase in signal intensity after Merck-3 treatment (Fig. 3A). Relative quantitation of the intensities in the Western blots showed a robust 70 to 85% reduction in sAPPβ relative to vehicle at all time points (p < 0.001; n = 4 mice in each group; Tukey-Kramer HSD). A modest but consistent increase in sAPP of 10 to 70%, relative to vehicle, was observed after treatment with Merck-3 (p < 0.05; Student's t test) (Fig. 3B). Brain sAPP showed a tendency to recover toward baseline at the 14-day time point, whereas brain sAPPβ remained inhibited at all time points.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Reduction of brain sAPPβ is associated with brain sAPP elevation after i.c.v. infusion with Merck-3. A, Western blots of sAPPβ and sAPP from brain homogenates in animals infused with vehicle (Veh) for 14 days or Merck-3 at 7.5 mkd for 1, 7, or 14 days of infusion. Equal amounts of protein were loaded in each lane, and each lane is a sample from a different animal. B, relative quantitation of gel intensities for sAPPβ and sAPP in A. sAPPβ levels were significantly reduced at 1, 7, and 14 days after Merck-3 infusion relative to vehicle-treated animals (1 day, 83 ± 1%; 7 day, 85 ± 5%; 14 day, 70 ± 7%; n = 4 animals each; p < 0.001, Tukey-Kramer HSD). Mean ± S.E.M. are shown (vehicle, n = 6 animals and n = 4 animals each at 1, 7, and 14 days of treatment with Merck-3). C, ELISA analysis of sAPPβ levels after treatment with 7.5 mkd Merck-3 for 1, 7, and 14 days, respectively. sAPPβ levels from untreated and Merck-3-treated animals were normalized to vehicle-treated animals. Data from n = 4 different experiments (n = 47 mice) are plotted together and represented by four different symbols: filled circles, filled squares, open circles, and open squares. There was a significant reduction of sAPPβ at 1 day (48 ± 7%; p < 0.001), 7 days (50 ± 9%; p < 0.001) and 14 days (71 ± 8%; p < 0.01), respectively, after Merck-3 treatment (via Tukey-Kramer HSD). D, ELISA analysis of sAPP levels after treatment with 7.5 mkd Merck-3 for 1, 7, or 14 days, respectively, as described in C. sAPP levels from untreated and Merck-3-treated animals were normalized to vehicle-treated levels. There was a significant elevation of sAPP at 1 day (118 ± 33%; p < 0.001) and at 7 days (82 ± 22%; p < 0.01), respectively, after Merck-3 treatment (via Tukey-Kramer HSD). In C and D, there is one animal that falls outside the whisker, where the whiskers extend from the ends of the box to the outermost data point in the upper end that is computed as "upper quartile + 1.5 * (interquartile range)" and in the lower end by "lower quartile–1.5 * (interquartile range)", where interquartile range is the difference between the quartiles. Thus, based on these calculations, this animal seems to be an outlier in the data set, but with no effect on the overall result.

The reduction of brain Aβ and sAPPβ with Merck-3 could also be due to a decrease in expression of BACE or APP in the brain, whereas the elevation in sAPP species could be due to an increase in the expression of ADAM10 or ADAM17, the putative -secretase enzymes. Brain mRNA expression levels for human APP, murine APP, BACE, ADAM10, and ADAM17 were assessed in mice treated with Merck-3 by real-time polymerase chain reaction (Supplemental Fig. 2). We observed a transient 1.5- to 2-fold increase in BACE1 and murine-APP mRNA levels at day 1 and 7, which declined back to levels similar to vehicle-treated animals, after Merck-3 treatment. There was no significant change in expression levels of human-APP, ADAM10, or ADAM17 at any of these time points. Thus, neither the brain Aβ nor sAPPβ lowering or sAPP elevation, with Merck-3 is due to a reduction in brain BACE or APP expression or to an up-regulation of ADAM10 or ADAM17 levels, respectively. These results confirm that inhibition of BACE1 leads to a decrease in brain sAPPβ production and an increase in brain sAPP secretion.

Neuregulin 1 Full-Length Protein Is Elevated in P15 Homozygote (–/–) and Heterozygote (+/–)-BACE1 KO mice compared with (+/+) Mice but Not in P30 or Aged Homozygote (–/–) Mice. Recent studies suggest that NRG-1, a protein that mediates juxtacrine-signaling between axons and Schwann cells and regulates axon myelination (Nave and Salzer, 2006), is a putative substrate for BACE1 (Hu et al., 2006; Willem et al., 2006). A reduction in NRG-1 processing was observed in young BACE1 KO mice (–/–), as evidenced by an increase in full-length NRG-1 protein levels (Willem et al., 2006) and a decrease in the N-terminal fragment of NRG-1 (Hu et al., 2006). BACE1 KO mice display a reduction in myelin thickness in peripheral nerves (Hu et al., 2006; Willem et al., 2006) and in the central nervous system (Hu et al., 2006). Consequently, it has been suggested that the decline in myelination during development in BACE1 KO mice, may be due to a reduction in NRG-1 processing and raises questions regarding the in vivo consequences of prolonged BACE1 inhibition on these parameters.

Before testing the effect of in vivo pharmacological inhibition of BACE1 on NRG-1 processing, we examined the consequences of gene dosage of BACE1 on BACE1 protein and NRG-1 processing in young mice, and we also tested whether the defect in NRG-1 processing was persistent in aged BACE1 KO or restricted to an early developmental period. We observed a gene dosage-dependent decrease in BACE1 protein in the BACE1 KO mice and cleavage products of APP (Supplemental Fig. 3). In the BACE1 KO (–/–) mice, BACE1 protein was absent, whereas (+/–) mice show intermediate levels compared with (+/+) mice. Likewise c99, the C-terminal fragment derived after BACE1 cleavage of APP, and sAPPβ, the N-terminal fragment of APP, were absent in the (–/–) mice compared with the (+/+) mice, whereas (+/–) mice show diminished levels but closer to levels in (+/+) animals (Cai et al., 2001; Luo et al., 2001) (Supplemental Fig. 3A). BACE1 KO (–/–) mice also show a complete lack of endogenous murine Aβ40 production, whereas the BACE1 (+/–) mice show 20% lower brain Aβ40 levels compared with the (+/+) mice (Supplemental Fig. 3B). Thus, an 50% drop in BACE1 protein levels only leads to 20% drop in brain Aβ40 levels in 3- to 4-month-old BACE1 KO mice, suggesting a nonlinear relationship of BACE activity on in vivo APP processing. Finally, an age-dependent decrease in both BACE1 protein levels (similar to Willem et al., 2006) and ex vivo BACE1 activity was observed in APP-YAC mice (Supplemental Fig. 3, C and D). Although the age-dependent decrease in BACE1 activity levels was correlated with a decline in BACE1 protein levels, BACE1 activity normalized to BACE1 protein levels were similar among the different ages (data not shown). These results suggest an age-dependent decrease in BACE1 protein levels and prompted us to examine the effect of age on NRG-1 processing in BACE1 KO mice.

Previous studies have shown that full-length NRG-1 protein levels were elevated in brains from BACE1 KO (–/–) mice compared with (+/+) animals from postnatal day 5 mice (Willem et al., 2006) and up to 2 months of age (Hu et al., 2006). Therefore, we first examined the gene dosage of BACE1 protein in brains from young (15 and 30 days old; P15 and P30) (–/–), (+/–), and (+/+) BACE1 KO mice and aged (2-year-old) (–/–) and (+/+) mice [unfortunately aged (+/–) mice were not available]. BACE1 KO (–/–) mice showed no detectable BACE1 protein in brain extracts from all three ages (Fig. 4, A–F). BACE1 KO (+/–) mice showed elevated intermediate levels of BACE1 protein, in the P15 and P30 animals, compared with (+/+) and (–/–) mice (p < 0.01; Tukey-Kramer HSD) (Fig. 4, A–D).

View larger version (49K): [in this window] [in a new window]   Fig. 4. Neuregulin1 full-length and N-terminal fragment are altered in P15 BACE1 KO (–/–) and (+/–) mice but not in P30 or 2-year-old mice brains. A, comparison of BACE1, NRG-1, NRG-NTF, MBP, and β-tubulin protein levels in brains from postnatal 15-day-old wild-type (+/+), heterozygote (+/–), and homozygote (–/–) BACE1 KO mice. Each lane represents a different animal. The BACE1 (–/–) mice show no detectable signal at 67 kDa, confirming a lack of BACE1 protein, whereas (+/–) showed intermediate signal compared with (+/+) mice. NRG-1 full-length protein (130 kDa) was significantly reduced in BACE1 (+/+) mice compared with BACE1 (+/–) and (–/–) mice. NRG-NTF showed modest decline in (+/+) mice compared with BACE1 (+/–) and (–/–) mice. MBP levels also showed a slight decline in (+/+) mice compared with BACE1 (+/–) and (–/–) mice. β-Tubulin levels were similar among the three genotypes of BACE1KO mice (gel loading control). B, Relative intensity quantification of BACE1, NRG-1 full-length protein, NRG-NTF, MBP, and tubulin in Western blots from A (mean ± S.E.M. of n = 3 mice). BACE1 protein is significantly reduced in (–/–) and (+/–) mice compared with (+/+) mice (p < 0.001 and p < 0.01, respectively; Tukey-Kramer HSD). Full-length NRG-1 protein is significantly higher in BACE1 (+/–) and (–/–) mice compared with (+/+) mice (p < 0.01; Tukey-Kramer HSD). NRG-NTF and MBP showed modest decline in (+/+) mice compared with (+/–) and (–/–) mice (p < 0.05; Tukey-Kramer HSD). Tubulin levels were not different among all the three genotypes. C, comparison of BACE1, NRG-1, NRG-NTF, MBP, and β-tubulin levels in brains from postnatal 30-day-old wild-type (+/+), heterozygote (+/–), and homozygote (–/–) BACE1 KO mice. Each lane represents a different animal. D, relative intensity quantification of BACE1, NRG-1 full-length protein, NRG-NTF, MBP, and tubulin in Western blots from C (mean ± S.E.M. of n = 3 mice). The BACE1 (–/–) mice show no detectable signal at 67 kDa, whereas (+/–) showed intermediate signal compared with (+/+) mice. NRG-1 full-length protein, NRG-NTF, and MBP levels were not different between BACE1 KO (+/+) mice compared with BACE1 (+/–) and (–/–) mice. Tubulin levels were similar among the three genotypes of BACE1KO mice (gel loading control). E, comparison of BACE1, NRG-1, NRG-NTF, MBP, and β-tubulin protein levels in brains from 2-year-old WT (+/+) and homozygote (–/–) BACE1 KO mice (+/–mice were not available). Each lane represents a different animal. F, relative intensity quantification of BACE1, NRG-1, NRG-NTF, MBP, and tubulin in Western blots from E (mean ± range of n = 2 mice). The BACE1 protein is reduced in (–/–) compared with (+/+) mice. NRG-1, NRG-NTF, MBP, and tubulin were not different between BACE (+/+) mice and (–/–) mice.

MBP levels were modestly reduced in BACE1 (+/+) mice compared with (+/–) and (–/–) mice in P15 mice, but no significant differences were observed among the genotypes in the P30 mice (Fig. 4, A–D). Finally, 2-year-old BACE1 (–/–) mice showed no detectable difference in MBP levels compared with (+/+) animals (Fig. 4, E and F). Thus, there was no evidence for long-term effects on brain MBP levels in the BACE1 KO mice.

Taken together, these results suggest that mature and older BACE1 KO animals do not show differences in brain NRG-1 processing like that observed in young P15 animals. Thus, BACE1-dependent processing of NRG-1 maybe more active in early development, and these effects may be compensated for with age in these mice. An alternate interpretation is that cleavage of NRG-1 is more robust in younger animals as evidenced by reduced levels of full-length NRG-1, whereas with increasing age NRG-1 cleavage declines to levels where no significant differences are observed between the BACE1 (+/+) and (–/–) animals.

Brain Neuregulin-1 Processing Is Unchanged after 1-Week i.c.v. Infusion with BACE Inhibitor Merck-3. We next examined the consequences of subchronic inhibition of BACE1, with Merck-3, on brain NRG-1 and its consequences on MBP levels in adult mice as a model to understand possible implications of BACE inhibition in adult humans. APP-YAC mice (4–6 months of age) were treated with Merck-3 via direct i.c.v. infusion at 7.5 mkd for 1 week (vehicle, n = 5 animals; Merck-3, n = 4 animals). BACE1 inhibition with Merck-3 led to a significant decrease in brain Aβ40 (36% decline; p < 0.05), Aβ42 (60% decline; p < 0.001) and sAPPβ (70% decline; p < 0.05), compared with vehicle-treated animals, confirming a robust in vivo inhibition of BACE1 (Fig. 5, A–C). In this same experiment, brain sAPP was elevated 53% (p < 0.05) (Fig. 5D). In Merck-3-treated animals, there were no significant changes in brain BACE1, full-length NRG-1 levels, N-terminal fragment of NRG-1, or MBP levels, compared with vehicle-treated animals (Fig. 5, E and F). Similar results were obtained in n = 2 independent experiments. These results suggest that robust inhibition of BACE1 in adult mice, which led to significant inhibition of brain APP processing, was not associated with any changes in NRG-1 processing in vivo.

View larger version (31K): [in this window] [in a new window]   Fig. 5. i.c.v. infusion with BACE1 inhibitor Merck-3 in adult mice led to no changes in brain neuregulin-1 processing, although APP processing was significantly inhibited. A, brain Aβ40 levels were significantly lowered in mice treated with 7.5 mkd Merck-3 (n = 4 mice) via i.c.v. infusion for 1 week compared with vehicle (n = 5 mice) (p < 0.05; Student's t test). B, brain Aβ42 levels were significantly lowered in mice treated with 7.5 mkd Merck-3 via i.c.v. infusion for 1 week (p < 0.001; Student's t test). C, brain sAPPβ levels were significantly lower in mice treated with 7.5 mkd Merck-3 for 1 week (p < 0.05; Student's t test). D, brain sAPP levels were significantly elevated in mice treated with 7.5 mkd Merck-3 for 1 week (p < 0.05; Student's t test). E, Western blots of brain homogenates from APP-YAC mice after i.c.v. infusion with vehicle (V) or Merck-3 (M3) at 7.5 mkd for 1 week, probed for BACE1, NRG-1, NRG-NTF, MBP, and β-tubulin from a subset of animals shown in A to D (each lane is a different mouse). F, relative intensity quantification of BACE1, NRG-1, MBP, and tubulin in Western blots from E (mean ± S.E.M. of n = 3 mice). There was no significant difference in BACE1, NRG-1, MBP, and tubulin levels between vehicle- and Merck-3-treated animals.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we have shown that direct intraventricular administration of Merck-3, a potent BACE1 inhibitor, led to both time- and dose-dependent lowering of brain Aβ40 and Aβ42 that could be sustained for up to 7 to 14 days of treatment. The decline in brain Aβ species was correlated with a decrease in brain sAPPβ, the N-terminal fragment of APP after BACE cleavage, thus confirming direct BACE1 inhibition. Concurrently, BACE1 inhibition led to a modest increase in brain sAPP, confirming that BACE1 and -secretase enzymes compete for cleavage of APP in vivo. Consistent with previous reports (Hu et al., 2006; Willem et al., 2006), an increase in full-length NRG-1 levels was observed in young P15 BACE1 KO (–/–) mice compared with (+/+) mice. Interestingly, we report first observations where (+/–) BACE1 KO mice displayed a similar increase in full-length NRG-1 like the (–/–) mice, and they lacked BACE1 gene dosage on NRG-1, compared with (+/+) mice. In contrast, there was a lack of a correlated decline in the N-terminal fragment of NRG-1 in the P15 BACE1 KO mice. In addition, 1-month-old P30 and 2-year-old BACE1 KO (–/–) mice and adult mice treated with a BACE1 inhibitor show no change in steady-state levels of brain full-length NRG-1 or NRG-NTF. Thus, in vivo BACE1 inhibition with Merck-3 in adult animals, which led to a significant decrease in brain APP processing, produced no measurable effect on NRG-1 processing. Taken together, these results suggest that pharmacological inhibition of BACE1 in adult animals can significantly lower brain Aβ levels and increase -secretase processing of APP without adversely affecting NRG-1 processing.

BACE1 is the major β-secretase in the brain that was identified and cloned using a variety of strategies (Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999; Lin et al., 2000). Although BACE1 inhibitor development has been challenging due to the large size and presence of numerous charged residues in the active site (Hong et al., 2000), numerous peptidomimetic BACE inhibitors have been developed (Baxter and Reitz, 2005; Ghosh et al., 2005). Although the first potent BACE1 inhibitor (Statine-val) was a peptidic inhibitor with a noncleavable statine and valine residue (IC₅₀ 30 nM) (Sinha et al., 1999), the subsequent statine-based inhibitors (Hong et al., 2000; Ghosh et al., 2001; Hu et al., 2004) had a variety of modifications, including hydroxyethylene isosteres (Hong et al., 2002; Hom et al., 2004), hydroxyethylamine isosteres (Tamamura et al., 2003; Stachel et al., 2004), hydroxymethylcarbonyl isosteres (Shuto et al., 2003; Kimura et al., 2004, 2005), and aminoethylene tetrahedral isosteres (Yang et al., 2006). Most of these transition state inhibitors had in vitro activity on BACE1, but many failed to work in cell culture systems or in vivo.

Merck-3, a hydoxyethylamine isostere, had an in vitro BACE1 IC₅₀ of 15 nM, and it showed robust lowering of secreted Aβ40 from brain slice cultures (IC₅₀ 30 nM). When administered peripherally via i.v. injection, no brain Aβ lowering was observed because it had poor apparent permeability and was a P-gp substrate. Central nervous system drugs typically require apparent permeabilities greater than 15 x 10^–6 cm/s and a P-gp transport ratio of <2.5 (Mahar Doan et al., 2002). When Merck-3 was introduced directly into the left lateral ventricle, a robust lowering of brain Aβ40 and Aβ42 in both brain hemispheres was observed. Merck-3 also led to a time- and dose-dependent lowering of brain sAPPβ, Aβ40, and Aβ42. Although brain Aβ40 and Aβ42 showed good correlation within a brain hemisphere, there was also excellent correlation of Aβ40 and 42 levels between the left and right brain hemisphere. The robust brain Aβ lowering is not probably due to nonspecific cellular toxicity of the surgery or to Merck-3, because animals infused with vehicle for 14 days showed no Aβ lowering compared with untreated animals; there was no difference in survival or gross behavioral changes between vehicle- and Merck-3-treated animals; a modest but consistent elevation of brain sAPP levels was observed, indicating lack of toxicity; and finally, brain mRNA expression studies suggest that brain Aβ lowering with Merck-3 is not due to a reduction of brain BACE or human-APP expression or to an up-regulation of ADAM10 or ADAM17. Therefore, we conclude that if a potent BACE1 inhibitor can be delivered past the blood-brain barrier, a sustained lowering of brain Aβ species can be achieved.

Although Merck-3 is a potent inhibitor of BACE1, it can also inhibit BACE2, with an IC₅₀ value of 300 nM (Pietrak et al., 2005). Thus, both BACE1 and BACE2 are probably inhibited at the concentrations of Merck-3 achieved via i.c.v. infusion. Although, BACE1 knockdown in cell cultures (Basi et al., 2003; Kao et al., 2004) or in mice (Singer et al., 2005) can lower sAPPβ production, BACE2 knockdown can actually increase sAPPβ and Aβ secretion, whereas simultaneous knockdown of BACE1 and BACE2 may produce no effects on secreted Aβ (Basi et al., 2003). Thus, BACE isoforms may compete for APP processing in some cell lines and reduction in cleavage at position 19 or 20 by BACE2 (Yan et al., 2001) could enhance cleavage at position 1 of Aβ sequence by BACE1 (Basi et al., 2003). Merck-3 led to robust lowering of brain sAPPβ while producing a significant elevation of brain sAPP levels, consistent with results from primary neurons from the BACE1 KO mice (Cai et al., 2001), and Tg2576 mice crossed with the BACE1 KO mice (Ohno et al., 2004; Laird et al., 2005). These observations, together with the poor brain expression of BACE2 (Bennett et al., 2000), strongly support the idea that BACE1 is the predominant β-site cleavage enzyme in brains of mice and that inhibition of BACE1 can effectively lower brain Aβ production.

The reduction of brain sAPPβ and associated elevation of brain sAPP may have beneficial effects because sAPP is suggested to have putative neuroprotective functions (Furukawa et al., 1996). In previous studies, it has been shown that activation of the M1-cholinergic receptors (Nitsch et al., 1992; Caccamo et al., 2006) and serotonin receptors (Nitsch et al., 1996) led to increased -secretase processing of APP, probably via protein kinase C activation (Skovronsky et al., 2000). Direct intracortical injection of PKC activators in mice showed an acute lowering of brain Aβ and sAPPβ, but no change in sAPP up to 6 h after injection (however, see Rossner et al., 2000; Savage et al., 1998). The rapid lowering of brain Aβ and sAPPβ are consistent with the short half-lives of 1 to 2 h estimated for brain Aβ and c99 (Savage et al., 1998; Cirrito et al., 2003). Mice overexpressing the -secretase enzyme ADAM10, crossed with the APPV717I transgenic mice, showed reduced Aβ production and amyloid plaque burden. In contrast, coexpression of dominant-negative mutant of ADAM10 with APPV717I enhanced amyloid pathology in double transgenic mice (Postina et al., 2004). Taken together, these results suggest that BACE1 and -secretase enzymes can compete for APP processing and that BACE1 inhibition can indirectly enhance -secretase processing of APP.

Our results are consistent with previous studies in which intraperitoneal administration of a hydroxyethylene isostere inhibitor conjugated to a peptide carrier (Chang et al., 2004) led to a dose- and time-dependent reduction of plasma and brain Aβ40. Intrahippocampal injection of a different BACE1 inhibitor (cell IC₅₀ 10 µM) showed in vivo lowering of brain Aβ40, Aβ42, and C99, but no change in C83 was observed in mice (Asai et al., 2006). More recently, a poorly brain penetrant BACE1 inhibitor when coadministered with a P-gp inhibitor lowered both brain Aβ40 and Aβ42, and it was accompanied by an increase in brain sAPP (Hussain et al., 2007). However, these previous studies have not addressed the impact of BACE1 inhibition on brain NRG-1 processing.

Neuregulin 1 type III (NRG-1) is a double transmembrane protein expressed on axonal membranes and cleaved by metalloproteases. The N-terminal membrane-bound fragment of NRG-1 can function as a juxtacrine-signaling molecule that regulates axon myelination via activation of ErbB receptors on Schwann cells (Nave and Salzer 2006). Recent studies have suggested that BACE1 regulates NRG-1 processing and signaling during development and thereby affects early myelination in the nervous system (Hu et al., 2006; Willem et al., 2006). BACE1 KO mice showed defects in myelination, as measured by an increase in G-ratio or myelin thickness in the sciatic nerves (Hu et al., 2006; Willem et al., 2006). Myelin thickness seems to increase gradually with age in BACE1 KO mice, change in G-ratio of 0.75 to 0.95 at P5 to P17 days to 0.70 to 0.85 in the adult (6–8 weeks of age), whereas the WT mice exhibit G-ratio of 0.60 to 0.80 over this age (Willem et al., 2006). These results suggest that downstream signaling of NRG-1, after BACE1 cleavage, maybe a critical event during myelination in early development.

An increase in full-length NRG-1 levels in the brain was observed in young P5 mice (Willem et al., 2006), but this study did not examine changes in the N-terminal fragment of NRG-1. Proximal studies by Hu et al. (2006) showed an increase in full-length NRG-1 levels in brain along with a reduction in the N-terminal fragment of NRG-1 in young (P30–P60) BACE1 KO (–/–) mice. These results suggested that BACE1 knockdown maybe accompanied by a decrease in in vivo processing of NRG-1 in young mice. However, these previous studies did not examine the affect of BACE1 gene dosage and older ages on NRG-1 processing.

The results in this study confirmed an increase in full-length NRG-1 protein in young P15 BACE1 KO (–/–) mice compared with WT mice, suggesting reduced NRG-1 processing. Although BACE1 protein levels were halved, an identical increase in full-length NRG-1, equivalent to that of BACE1 (–/–) mice, was observed in BACE1 (+/–) mice compared with WT (+/+) mice. These results suggest a decrease in NRG-1 processing in both BACE1 (+/–) and (–/–) mice. These results are in contrast to the effect of BACE1 knock-down on APP processing, where BACE1 (+/–) mice showed only 20% reduction in brain Aβ40 levels compared with (+/+) mice, whereas (–/–) mice showed an almost complete reduction in brain Aβ levels (Supplemental Fig. 3). Thus, neither APP nor NRG-1 processing shows precise gene dosage dependence with BACE1 knockdown, raising questions regarding the exact physiological substrate(s) for BACE1 in vivo. In contrast, BACE1-dependent processing of substrates may not be solely regulated by absolute expression levels, but by differential colocalization with substrates and intracellular localization similar to that suggested in other reports (Sinha and Lieberburg, 1999; Greenfield et al., 2000; Pasternak et al., 2004). We envision that future studies could examine the affect of BACE1 gene dosage on myelination in parallel with NRG-1 and other substrates.

Although an increase in full-length NRG-1 was observed in P15 BACE1 (–/–) and (+/–) mice, we observed a modest decline in the N-terminal fragment of NRG-1 in BACE1 (+/+) mice compared with BACE1 (+/–) and (–/–) mice in contrast to a previous report (Hu et al., 2006). The possible reasons for the differences between these studies are presently unknown, but they could be due to difference in genetic background or difference in the locus where BACE1 was knocked out in these mice. Mice used in our studies were rederived from that generated in Cai et al. (2001) and back-crossed to the C57BL/6 mice. Thus, although mice used in our studies and Hu et al. (2006) seem to be of the same origin, backcrossing to a pure genetic background may have led to the differences observed.

BACE1 protein levels and activity are 2- to 3-fold higher (our results) and manyfold higher (Willem et al., 2006) in mice from ages of P0 to P15 compared with mice P30 and older, thus motivating experiments to examine the age dependence of NRG-1 processing. Examination of NRG-1 processing in P30 BACE1 KO mice revealed no differences in steady-state levels of either full-length NRG-1 or NRG-NTF among all three genotypes in our studies. Finally, 2-year-old BACE1 KO (–/–) mice showed no change in full-length NRG-1 or NRG-NTF levels compared with WT mice. Our results are consistent with those of both Willem et al. (2006) and Hu et al. (2006) in that young BACE1 KO mice show an increase in full-length NRG-1 compared with (+/+) mice. Although NRG-NTF was modestly elevated in (+/+) mice in Hu et al. (2006), we observed a modest decline in NRG-NTF in P15 mice, and no differences were seen in P30 and 2-year-old mice, whereas Willem et al. (2006) did not examine NRG-NTF in their studies. Thus, our results point to a transient effect of BACE1 knockdown on NRG-1 processing in mice, and they suggest differences in NRG-1 processing only in early development. The age-dependent decrease in BACE1 expression could explain the transient effect on NRG-1 processing, and it can also account for the lack of significant differences in NRG-1 and NRG-NTF between BACE1 KO and (+/+) mice at ages P30 and older. Thus, it seems that with age, NRG-1 processing declines in (+/+) mice to levels equivalent to that in the BACE1 (–/–) mice, and this might explain the lack of differences in NRG-1 processing in older ages.

Finally, inhibition of BACE1 over the course of 1 week in adult APP-YAC mice, via direct i.c.v. infusion of Merck-3, did not alter full-length NRG-1 or NRG-NTF levels or MBP levels compared with vehicle-treated mice, although a robust inhibition of APP processing and Aβ production was observed. These results suggest that acute inhibition of BACE1 in adult mice is without significant impact on NRG-1 or NRG-NTF, although APP processing is significantly altered. The lack of an effect of Merck-3 on NRG-1 processing is consistent with our observations that there was minimal impact of BACE1 knockdown on NRG-1 processing in older mice. However, this cannot be predicted a priori because pharmacological inhibition with a compound can significantly change the steady-state processing of substrate as in the case of APP. In conclusion, although we have consistently observed that in vivo BACE1 inhibition leads to significant decline in brain sAPPβ and Aβ production, steady-state NRG-1 processing was unaffected. These results suggest that BACE1 inhibition may be devoid of adverse effects due to altered NRG-1 processing in adult humans. Recent studies indicate that NRG-1 can directly affect glutamatergic and GABAergic synapse maturation and transmission in vivo (Li et al., 2007; Woo et al., 2007). Therefore, the interplay of NRG-1 and BACE1 on synaptic function and the impact of BACE1 inhibition should motivate further investigation.

In conclusion, our results support BACE1 inhibition as a viable target to lower brain Aβ40 and Aβ42 and to enhance the nonamyloidogenic processing of APP via -secretase cleavage without adversely affecting NRG-1 downstream events. In recent studies, there have been a number of promising reports of acute lowering of brain Aβ with BACE inhibitors after peripheral administration, but these inhibitors lack the properties required for sustained in vivo efficacy (Stachel et al., 2006; Hussain et al., 2007; Stanton et al., 2007). Further optimization of BACE inhibitors for improved potency, physical, and pharmacokinetic properties would allow better understanding of the therapeutic effects and potential drawbacks of long-term BACE inhibition.

	Acknowledgements

 
We thank Jennifer Shapiro and Mark Stiteler for help in the neuregulin experiments and Dr. Thomas William Mitchell, Jim Destefano, Sandra Veggian, Jeanine Hange, and Ryan Desautels (Merck Research Laboratories, West Point Laboratory Animal Resources team) for technical support in the animal surgeries. Finally, we gratefully acknowledge the extensive comments of the reviewers.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.130039.

ABBREVIATIONS: AD, Alzheimer's disease; BACE, β-secretase; APP, amyloid precursor protein; sAPP, soluble amyloid precursor protein; CTF, C-terminal fragment; Aβ, amyloid β peptide (those mentioned herein consist of 40 or 42 amino acids); KO, knockout; NRG, neuregulin-1; P, postnatal day; WT, wild type; ELISA, enzyme-linked immunosorbent assay; AP, alkaline phosphatase; mkd, milligrams per kilogram per day; PBS, phosphate-buffered saline; MBP, myelin basic protein; HSD, honestly significant difference; NTF, N-terminal fragment; P-gp, P-glycoprotein; ANOVA, analysis of variance.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

¹ Current affiliation: Department of Oncology, GlaxoSmithKline, Collegeville, Pennsylvania.

Address correspondence to: Dr. Adam J. Simon, Department of Alzheimer's Research, WP 26A-2000, Merck Research Laboratories, 770, Sumneytown Pike, West Point, PA 19486. E-mail: adam_simon{at}merck.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Asai M, Hattori C, Iwata N, Saido TC, Sasagawa N, Szabo B, Hashimoto Y, Maruyama K, Tanuma S, Kiso Y, et al. (2006) The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice. J Neurochem 96: 533–540.[CrossRef][Medline]

Basi G, Frigon N, Barbour R, Doan T, Gordon G, McConlogue L, Sinha S, and Zeller M (2003) Antagonistic effects of beta-site amyloid precursor protein-cleaving enzymes 1 and 2 on β-amyloid peptide production in cells. J Biol Chem 278: 31512–31520.[Abstract/Free Full Text]

Baxter EW and Reitz AB (2005) BACE inhibitors for the treatment of Alzheimer's disease, in Annual Reports in Medicinal Chemistry, Vol. 40, pp. 35–48, Elsevier, New York.[Medline]

Bennett BD, Babu-Khan S, Loeloff R, Louis JC, Curran E, Citron M, and Vassar R (2000) Expression analysis of BACE2 in brain and peripheral tissues. J Biol Chem 275: 20647–20651.[Abstract/Free Full Text]

Best JD, Jay MT, Otu F, Ma J, Nadin A, Ellis S, Lewis HD, Pattison C, Reilly M, Harrison T, et al. (2005) Quantitative measurement of changes in amyloid-β(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide]. J Pharmacol Exp Ther 313: 902–908.[Abstract/Free Full Text]

Bodendorf U, Danner S, Fischer F, Stefani M, Sturchler-Pierrat C, Wiederhold KH, Staufenbiel M, and Paganetti P (2002) Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid. J Neurochem 80: 799–806.[CrossRef][Medline]

Braak H and Braak E (1991) Neuropathological staging of Alzheimer-related changes. Acta Neuropathol 82: 239–259.[CrossRef][Medline]

Caccamo A, Oddo S, Billings LM, Green KN, Martinez-Coria H, Fisher A, and LaFerla FM (2006) M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron 49: 671–682.[CrossRef][Medline]

Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, and Wong PC (2001) BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4: 233–234.[CrossRef][Medline]

Chang WP, Koelsch G, Wong S, Downs D, Da H, Weerasena V, Gordon B, Devasamudram T, Bilcer G, Ghosh AK, et al. (2004) In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors. J Neurochem 89: 1409–1416.[CrossRef][Medline]

Cirrito JR, May PC, O'Dell MA, Taylor JW, Parsadanian M, Cramer JW, Audia JE, Nissen JS, Bales KR, Paul SM, et al. (2003) In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. J Neurosci 23: 8844–8853.[Abstract/Free Full Text]

Citron M (2004) Strategies for disease modification in Alzheimer's disease. Nat Rev Neurosci 5: 677–685.[CrossRef][Medline]

Dominguez D, Tournoy J, Hartmann D, Huth T, Cryns K, Deforce S, Serneels L, Camacho IE, Marjaux E, Craessaerts K, et al. (2005) Phenotypic and biochemical analyses of BACE1- and BACE2-deficient mice. J Biol Chem 280: 30797–30806.[Abstract/Free Full Text]

Furukawa K, Sopher BL, Rydel RE, Begley JG, Pham DG, Martin GM, Fox M, and Mattson MP (1996) Increased activity-regulating and neuroprotective efficacy of alpha-secretase-derived secreted amyloid precursor protein conferred by a C-terminal heparin-binding domain. J Neurochem 67: 1882–1896.[Medline]

Ghosh AK, Bilcer G, Harwood C, Kawahama R, Shin D, Hussain KA, Hong L, Loy JA, Nguyen C, Koelsch G, et al. (2001) Structure-based design: potent inhibitors of human brain memapsin 2 (beta-secretase). J Med Chem 44: 2865–2868.[CrossRef][Medline]

Ghosh AK, Kumaragurubaran N, and Tang J (2005) Recent developments of structure based beta-secretase inhibitors for Alzheimer's disease. Curr Top Med Chem 5: 1609–1622.[CrossRef][Medline]

Greenfield JP, Gross RS, Gouras GK, and Xu H (2000) Cellular and molecular basis of beta-amyloid precursor protein metabolism. Front Biosci 5: D72–D83.[Medline]

Hardy J and Selkoe DJ (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297: 353–356.[Abstract/Free Full Text]

Harrison SM, Harper AJ, Hawkins J, Duddy G, Grau E, Pugh PL, Winter PH, Shilliam CS, Hughes ZA, Dawson LA, et al. (2003) BACE1 (beta-secretase) transgenic and knockout mice: identification of neurochemical deficits and behavioral changes. Mol Cell Neurosci 24: 646–655.[CrossRef][Medline]

Hom RK, Gailunas AF, Mamo S, Fang LY, Tung JS, Walker DE, Davis D, Thorsett ED, Jewett NE, Moon JB, et al. (2004) Design and synthesis of hydroxyethylene-based peptidomimetic inhibitors of human beta-secretase. J Med Chem 47: 158–164.[CrossRef][Medline]

Hong L, Koelsch G, Lin X, Wu S, Terzyan S, Ghosh AK, Zhang XC, and Tang J (2000) Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. Science 290: 150–153.[Abstract/Free Full Text]

Hong L, Turner RT III, Koelsch G, Shin D, Ghosh AK, and Tang J (2002) Crystal structure of memapsin 2 (β-secretase) in complex with an inhibitor OM00–3. Biochemistry 41: 10963–10967.[CrossRef][Medline]

Hu B, Fan KY, Bridges K, Chopra R, Lovering F, Cole D, Zhou P, Ellingboe J, Jin G, Cowling R, et al. (2004) Synthesis and SAR of bis-statine based peptides as BACE 1 inhibitors. Bioorg Med Chem Lett 14: 3457–4560.[CrossRef][Medline]

Hussain I, Hawkins J, Harrison D, Hille C, Wayne G, Cutler L, Buck T, Walter D, Demont E, Howes C, et al. (2007) Oral administration of a potent and selective non-peptidic BACE-1 inhibitor decreases beta-cleavage of amyloid precursor protein and amyloid-beta production in vivo. J Neurochem 100: 802–809.[CrossRef][Medline]

Hussain I, Powell D, Howlett DR, Tew DG, Week TD, Chapman C, Gloger IS, Murphy KE, Southan CD, Ryan DM, et al. (1999) Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci 14: 419–427.[CrossRef][Medline]

Hu X, Hicks CW, He W, Wong P, Macklin WB, Trapp BD, and Yan R (2006) Bace1 modulates myelination in the central and peripheral nervous system. Nat Neurosci 9: 1520–1525.[CrossRef][Medline]

Ida N, Hartmann T, Pantel J, Schroder J, Zerfass R, Forstl H, Sandbrink R, Masters CL, and Beyreuther K (1996) Analysis of heterogeneous A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay. J Biol Chem 271: 22908–22914.[Abstract/Free Full Text]

Kao SC, Krichevsky AM, Kosik KS, and Tsai LH (2004) BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem 279: 1942–1949.[Abstract/Free Full Text]

Kawarabayashi T, Younkin LH, Saido TC, Shoji M, Ashe KH, and Younkin SG (2001) Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease. J Neurosci 21: 372–381.[Abstract/Free Full Text]

Kim KS, Miller DL, Sapienza VJ, Chen CJ, Bai C, Grundke-Iqbal I, Currie JR, and Wisniewski HM (1988) Production and characterization of monoclonal antibodies reactive to synthetic cerebrovascular amyloid peptide. Neurosci Res Commun 2: 121–130.

Kimura T, Shuto D, Hamada Y, Igawa N, Kasai S, Liu P, Hidaka K, Hamada T, Hayashi Y, and Kiso Y (2005) Design and synthesis of highly active Alzheimer's beta-secretase (BACE1) inhibitors, KMI-420 and KMI-429, with enhanced chemical stability. Bioorg Med Chem Lett 15: 211–215.[CrossRef][Medline]

Kimura T, Shuto D, Kasai S, Liu P, Hidaka K, Hamada T, Hayashi Y, Hattori C, Asai M, Kitazume S, et al. (2004) KMI-358 and KMI-370, highly potent and small-sized BACE1 inhibitors containing phenylnorstatine. Bioorg Med Chem Lett 14: 1527–1531.[CrossRef][Medline]

Laird FM, Cai H, Savonenko AV, Farah MH, He K, Melnikova T, Wen H, Chiang HC, Xu G, Koliatsos VE, et al. (2005) BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions. J Neurosci 25: 11693–11709.[Abstract/Free Full Text]

Lamb BT, Sisodia SS, Lawler AM, Slunt HH, Kitt CA, Kearns WG, Pearson PL, Price DL, and Gearhart JD (1993) Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice. Nat Genet 5: 22–30.[Medline]

LeBlanc AC, Koutroumanis M, and Goodyer CG (1998) Protein kinase C activation increases release of secreted amyloid precursor protein without decreasing Abeta production in human primary neuron cultures. J Neurosci 18: 2907–2913.[Abstract/Free Full Text]

Lee EB, Zhang B, Liu K, Greenbaum EA, Doms RW, Trojanowski JQ, and Lee VM (2005) BACE overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo. J Cell Biol 168: 291–302.[Abstract/Free Full Text]

Li B, Woo RS, Mei L, and Malinow R (2007) The neuregulin-1 receptor erbB4 controls glutamatergic synapse maturation and plasticity. Neuron 54: 583–597.[CrossRef][Medline]

Lin X, Koelsch G, Wu S, Downs D, Dashti A, and Tang J (2000) Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A 97: 1456–1460.[Abstract/Free Full Text]

Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, et al. (2001) Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4: 231–232.[CrossRef][Medline]

Mahar Doan KM, Humphreys JE, Webster LO, Wring SA, Shampine LJ, Serabjit-Singh CJ, Adkison KK, and Polli JW (2002) Passive permeability and P-glycoprotein-mediated efflux differentiate central nervous system (CNS) and non-CNS marketed drugs. J Pharmacol Exp Ther 303: 1029–1037.[Abstract/Free Full Text]

Mohajeri MH, Saini KD, and Nitsch RM (2004) Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology. J Neural Transm 111: 413–425.[CrossRef][Medline]

Naslund J, Haroutunian V, Mohs R, Davis KL, Davies P, Greengard P, and Buxbaum JD (2000) Correlation between elevated levels of amyloid beta peptide in the brain and cognitive decline. JAMA 283: 1571–1577.[Abstract/Free Full Text]

Nave KA and Salzer JL (2006) Axonal regulation of myelination by neuregulin 1. Curr Opin Neurobiol 16: 492–500.[CrossRef][Medline]

Nishitomi K, Sakaguchi G, Horikoshi Y, Gray AJ, Maeda M, Hirata-Fukae C, Becker AG, Hosono M, Sakaguchi I, Minami SS, et al. (2006) BACE1 inhibition reduces endogenous Abeta and alters APP processing in wild-type mice. J Neurochem 99: 1555–1563.[CrossRef][Medline]

Nitsch RM, Deng M, Growdon JH, and Wurtman RJ (1996) Serotonin 5-HT2a and 5-HT2c receptors stimulate amyloid precursor protein ectodomain secretion. J Biol Chem 271: 4188–4194.[Abstract/Free Full Text]

Nitsch RM, Slack BE, Wurtman RJ, and Growdon JH (1992) Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. Science 258: 304–307.[Abstract/Free Full Text]

Ohno M, Sametsky EA, Younkin LH, Oakley H, Younkin SG, Citron M, Vassar R, and Disterhoft JF (2004) BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer's disease. Neuron 41: 27–33.[CrossRef][Medline]

Pasternak SH, Callahan JW, and Mahuran DJ (2004) The role of the endosomal/lysosomal system in amyloid-beta production and the pathophysiology of Alzheimer's disease: reexamining the spatial paradox from a lysosomal perspective. J Alzheimers Dis 6: 53–65.[Medline]

Paxinos G and Franklin KBJ (2001) The Mouse Brain in Stereotaxic Coordinates, 2nd ed, Academic Press, San Diego.

Pietrak BL, Crouthamel MC, Tugusheva K, Lineberger JE, Xu M, DiMuzio JM, Steele T, Espeseth AS, Stachel SJ, Coburn CA, et al. (2005) Biochemical and cell-based assays for characterization of BACE-1 inhibitors. Anal Biochem 342: 144–151.[CrossRef][Medline]

Postina R, Schroeder A, Dewachter A, Bohl J, Schmitt U, Kojro E, Prinzen C, Endres K, Hiemke C, Blessing M, et al. (2004) A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest 113: 1456–1464.[CrossRef][Medline]

Roberds SL, Anderson J, Basi G, Bienkowski MJ, Branstetter DG, Chen KS, Freedman SB, Frigon NL, Games D, Hu K, et al. (2001) BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet 10: 1317–1324.[Abstract/Free Full Text]

Rossner S, Beck M, Stahl T, Mendla K, Schliebs R, and Bigl V (2000) Constitutive overactivation of protein kinase C in guinea pig brain increases alpha-secretory APP processing without decreasing beta-amyloid generation. Eur J Neurosci 12: 3191–3200.[CrossRef][Medline]

Sambamurti K, Greig NH, and Lahiri DK (2002) Advances in the cellular and molecular biology of the beta-amyloid protein in Alzheimer's disease. Neuromolecular Med 1: 1–31.[CrossRef][Medline]

Savage MJ, Trusko SP, Howland DS, Pinsker LR, Mistretta S, Reaume AG, Greenberg BD, Siman R, and Scott RW (1998) Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester. J Neurosci 18: 1743–1752.[Abstract/Free Full Text]

Scheff SW and Price DA (2003) Synaptic pathology in Alzheimer's disease: a review of ultrastructural studies. Neurobiol Aging 24: 1029–1046.[CrossRef][Medline]

Shi XP, Tugusheva K, Bruce JE, Lucka A, Chen-Dodson E, Hu B, Wu GX, Price E, Register RB, Lineberger J, et al. (2005) Novel mutations introduced at the beta-site of amyloid beta protein precursor enhance the production of amyloid beta peptide by BACE1 in vitro and in cells. J Alzheimers Dis 7: 139–148.[Medline]

Shuto D, Kasai S, Kimura T, Liu P, Hidaka K, Hamada T, Shibakawa S, Hayashi Y, Hattori C, Szabo B, et al. (2003) KMI-008, a novel beta-secretase inhibitor containing a hydroxymethylcarbonyl isostere as a transition-state mimic: design and synthesis of substrate-based octapeptides. Bioorg Med Chem Lett 13: 4273–4276.[CrossRef][Medline]

Singer O, Marr RA, Rockenstein E, Crews L, Coufal NG, Gage FH, Verma IM, and Masliah E (2005) Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model. Nat Neurosci 8: 1343–1349.[CrossRef][Medline]

Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan M, Dovey HF, Frigon N, Hong J, et al. (1999) Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402: 537–540.[CrossRef][Medline]

Sinha S and Lieberburg I (1999) Cellular mechanisms of beta-amyloid production and secretion. Proc Natl Acad Sci U S A 96: 11049–11053.[Abstract/Free Full Text]

Skovronsky DM, Moore DB, Milla ME, Doms RW, and Lee VM (2000) Protein kinase C-dependent -secretase competes with β-secretase for cleavage of amyloid-β precursor protein in the trans-Golgi network. J Biol Chem 275: 2568–2575.[Abstract/Free Full Text]

Stachel SJ, Coburn CA, Sankaranarayanan S, Price EA, Pietrak BL, Huang Q, Lineberger J, Espeseth AS, Jin L, Ellis J, et al. (2006) Macrocyclic inhibitors of beta-secretase: functional activity in an animal model. J Med Chem 49: 6147–6150.[CrossRef][Medline]

Stachel SJ, Coburn CA, Steele TG, Jones KG, Loutzenhiser EF, Gregro AR, Rajapakse HA, Lai MT, Crouthamel MC, Xu M, et al. (2004) Structure-based design of potent and selective cell-permeable inhibitors of human beta-secretase (BACE-1). J Med Chem 47: 6447–6450.[CrossRef][Medline]

Stanton MG, Stauffer SR, Gregro AR, Steinbeiser M, Nantermet P, Sankaranarayanan S, Price EA, Wu G, Crouthamel MC, Ellis J, et al. (2007) Discovery of isonicotinamide derived beta-secretase inhibitors: in vivo reduction of beta-amyloid. J Med Chem 50: 3431–3433.[CrossRef][Medline]

Tamamura H, Kato T, Otaka A, and Fujii N (2003) Synthesis of potent β-secretase inhibitors containing a hydroxyethylamine dipeptide isostere and their structure-activity relationship studies. Org Biomol Chem 1: 2468–2473.[CrossRef][Medline]

Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, and Katzman R (1991) Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30: 572–580.[CrossRef][Medline]

Trojanowski JQ, Shin RW, Schmidt ML, and Lee VM (1995) Relationship between plaques, tangles, and dystrophic processes in Alzheimer's disease. Neurobiol Aging 16: 335–340.[CrossRef][Medline]

Umbenhauer DR, Lankas GR, Pippert TR, Wise LD, Cartwright ME, Hall SJ, and Beare CM (1997) Identification of a P-glycoprotein-deficient subpopulation in the CF-1 mouse strain using a restriction fragment length polymorphism. Toxicol Appl Pharmacol 146: 88–94.[CrossRef][Medline]

Vassar R (2004) BACE1: the beta-secretase enzyme in Alzheimer's disease. J Mol Neurosci 23: 105–114.[CrossRef][Medline]

Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, et al. (1999) Beta-Secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286: 735–741.[Abstract/Free Full Text]

Willem M, Dewachter I, Smyth N, Van Dooren T, Borghgraef P, Haass C, and Van Leuven F (2004) β-Site amyloid precursor protein cleaving enzyme 1 increases amyloid deposition in brain parenchyma but reduces cerebrovascular amyloid angiopathy in aging BACExAPP[V717I] double-transgenic mice. Am J Pathol 165: 1621–1631.[Abstract/Free Full Text]

Willem M, Garratt AN, Novak B, Citron M, Kaufmann S, Rittger A, Destrooper B, Saftig P, Birchmeier C, and Haass C (2006) Control of peripheral nerve myelination by the beta-secretase BACE1. Science 314: 664–666.[Abstract/Free Full Text]

Woo RS, Li XM, Tao Y, Carpenter-Hyland E, Huang YZ, Weber J, Neiswender H, Dong XP, Wu J, Gassmann M, et al. (2007) Neuregulin-1 enhances depolarization-induced GABA release. Neuron 54: 599–610.[CrossRef][Medline]

Yang W, Lu W, Lu Y, Zhong M, Sun J, Thomas AE, Wilkinson JM, Fucini RV, Lam M, Randal M, et al. (2006) Aminoethylenes: a tetrahedral intermediate isostere yielding potent inhibitors of the aspartyl protease BACE-1. J Med Chem 49: 839–842.[CrossRef][Medline]

Yan R, Munzner JB, Shuck ME, and Bienkowski MJ (2001) BACE2 functions as an alternative alpha-secretase in cells. J Biol Chem 276: 34019–34027.[Abstract/Free Full Text]

Yan RQ, Bienkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM, Brashier JR, Stratman NC, Mathews WR, Buhl AE, et al. (1999) Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature 402: 533–537.[CrossRef][Medline]

Zheng H, Jiang M, Trumbauer ME, Sirinathsinghji DJ, Hopkins R, Smith DW, Heavens RP, Dawson GR, Boyce S, Conner MW, et al. (1995) beta-Amyloid precursor protein-deficient mice show reactive gliosis and decreased locomotor activity. Cell 81: 525–531.[CrossRef][Medline]

Zhu G, Wang D, Lin YH, McMahon T, Koo EH, and Messing RO (2001) Protein kinase C epsilon suppresses Abeta production and promotes activation of alpha-secretase. Biochem Biophys Res Commun 285: 997–1006.[CrossRef][Medline]

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