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NEUROPHARMACOLOGY
-Amyloid Profiles Studies of Interspecies Variation and Drug Action by Internally Standardized Immunoprecipitation/Mass SpectrometryPfizer Global Research and Development, Groton, Connecticut
Received October 2, 2006; accepted December 27, 2006.
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Abstract |
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-Amyloid peptides, tentatively regarded as the principal neurotoxins responsible for Alzheimer's Disease, make up a set of products that varies significantly among different biological systems. The full implications of this complexity and its variations have yet to be defined. In this work, A peptide populations were extracted from animal brain tissue or cell-conditioned media, immunoprecipitated with specific antibodies, and analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. 15N-Substituted A internal standards were added to gauge variations in the profile of captured peptides. Results from a range of species, including guinea pig, dog, rabbit, and wild-type and transgenic mice, showed that the A peptide population in each system was mainly determined by the species of origin of the amyloid precursor protein (APP) and not by the host tissue or cell line. The same method was used to gauge the effect on the A peptide profile of an inhibitor of 
-secretase, one of the two proteinases that excises A peptides from the precursor protein with different effects on specific peptides. Overall, the results demonstrate that the species of origin of the APP substrate dictates the outcome of APP processing to a greater extent than the origin of the processing enzymes, an important consideration in rationalizing the properties of different model systems.
peptides are tentatively regarded as the principal neurotoxins responsible for AD (Watson et al., 2005
) and are formed by sequential proteolytic events near the C terminus of the 770-residue APP. They are also the main components of amyloid plaques that are the classical biomarker of AD in post mortem brain. In the first step of pathological processing, the membrane-bound aspartic proteinase BACE cleaves APP after Met-671 to generate a C-terminal fragment called APP-C-terminal fragment .A species are liberated from this fragment by the intramembrane proteinase -secretase. Both secretases are seen as important drug targets, because the amyloid hypothesis of AD implies that blocking the production of A peptides will retard the disease (Gandy, 2005). The alternative 
-secretory pathway does not lead to A peptide production.
BACE is an atypical aspartic proteinase (Haniu et al., 2000), but it clearly belongs to that enzyme family and can be crystallized (Hong et al., 2000). -Secretase is a much more enigmatic target. It too is an aspartic-dependent proteinase, but of a specialized type adapted for regulated intramembrane proteolysis (Landman and Kim, 2004). Its proteolytic activity resides in a complex that includes PS and several other proteins (Edbauer et al., 2004; Chyung et al., 2005). -Secretase cleaves APP at various sites to produce A peptides of different lengths. Cleavage at the "-site" produces A1–40 and A1–42, the products that are widely attributed to play a major role in AD. These peptides oligomerize, aggregate, and ultimately accumulate in plaques (Citron et al., 1996). -Site cleavage may be preceded by action of -secretase at the "
site" located after Leu-720 of APP, which releases a 50-residue peptide known as AICD (amyloid intracellular domain) (Gao and Pimplikar, 2001). More recently, formation of a 46-residue fragment of APP has also been attributed to -secretase activity and termed the 
cleavage (Zhao et al., 2004).
Analysis of AD brain tissue and brain tissue from transgenic mice expressing human APP has detected a variety of different A peptides in extracellular amyloid deposits. In addition to A1–40 and A1–42, N-terminally and C-terminally truncated peptides and C-terminally elongated peptides have been reported. A peptides with an N-terminal Glu-3 or Glu-11 (Naslund et al., 1994), as well as peptides with pyroglutamyl N termini at positions 3 and 11 (Miravalle et al., 2005; Rufenacht et al., 2005), have been detected in amyloid plaques from human brain. Plaques from transgenic mice expressing both human APP and PS, as well as plaques from human AD brains, have been reported to contain A peptides 1 to 16 and 17 to 28 (Rufenacht et al., 2005). C-terminally truncated peptides, including A1–37, A1–38, and A1–39, have also been found in plaques from two different transgenic mouse lines (Lewis et al., 2004) and in cerebrospinal fluid of AD patients (Lewczuk et al., 2003). The biological function and disease relevance of the various A peptides is not understood, and the full complexity of processing, both of the precursor protein and A fragments, has yet to be resolved.
The production of A1–42 is favored by mutations of APP or PS linked to early-onset or familial forms of AD (Clark et al., 1995). Because A1–42 forms fibrils more readily than most other A peptides and is the major component of congophilic plaques in AD brain, the production of A1–42 is believed to favor progression of the disease. Thus, both the substrate sequence and mutations within the enzyme may influence the final profile of products from -secretase-catalyzed cleavage of APP, and subtle differences in these profiles may be highly significant.
These considerations led us to use IP followed by MALDI-TOF MS to observe A profiles from the brains of several preclinical species and from transgenic mice expressing mutant forms of human APP. Recently, several groups have reported the incorporation of internal standards into A samples subjected to MS, allowing quantitation of specific A peptides (Miravalle et al., 2005; Rufenacht et al., 2005). In this work, we incorporated multiple internal standards to monitor changes in several specific A peptides. The results indicated that the sequence of APP is the major factor governing which A peptides are produced by proteolytic processing and that the species identity of the host cell or tissue is a less important factor.
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Materials and Methods |
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Brain Extracts. Methods for handling animals were approved by the Animal Care and Use Committee. Animals were euthanized according to animal care guidelines, and the brains were removed and frozen in liquid N2. Brains were homogenized in a Polytron homogenizer using a 9-fold milliliters-to-grams ratio of ice-cold 0.2% DEA/0.05 M NaCl (Wong et al., 2004). The homogenate was incubated at 0°C for 3 h and then centrifuged at 135,000g for 1 h. Supernatants were collected and stored frozen at -80°C.
Immunoprecipitation from Brain Extract. Brain extract was adjusted to pH 8.5 by adding a 1:100 dilution of 2 M Tris-HCl, pH 8.5, after which the sample was treated with three-quarters of a tablet of Complete EDTA-Free Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN). Proteins that bound nonspecifically to beads were removed by treating the sample with 0.8 ml of a slurry of ImmunoPure Immobilized Protein A/G beads (Pierce Chemical) and incubating it overnight at 4°C with end-over-end rotation, after which it was centrifuged to remove the beads. Next, the supernatant was treated with isotopically substituted A standards (rPeptide, Bogart, GA), chosen to allow quantification of the relevant peptides. In specific experiments with animal models expressing human sequence A, one or two or all of the following 15N-substituted human standards were used in the amounts: 7 ng of A1–40N15, 3.5 ng of A1–42N15, and 3.5 ng of A1–43N15. In specific experiments with animal models expressing murine/rat sequence, 7 ng of A1–40N15-substituted murine/rat standard was added. Beads loaded with A-specific antibodies were then added to the sample (see above), and the samples were incubated with rotation for 24 h, after which the beads were separated from the rest of the sample by centrifugation. The beads were washed three times for 300 s each with 50 ml of ice-cold 0.2% DEA, 0.02 M Tris HCl, and 0.05 M NaCl, pH 8.5 and recovered from each wash by centrifugation at 1530g. They were then transferred to a 1.5-ml microcentrifuge tube and washed twice with 1.5 ml of ice-cold 0.02 M Tris HCl, 0.05 M NaCl, pH 8.0. Finally, the captured peptides were recovered by eluting them from the antibody beads with 50% CH3CN/0.1% trifluoroacetic acid, and the eluate was then neutralized with NH4OH.
Operational Scale of Immunoprecipitation with Different A Sources. The intention of different scaling was to obtain about the same amount of A for mass spectrometry from each IP experiment. With 2-week-old male Hartley guinea pigs (Charles River, Laboratories, Wilmington, MA), 30 ml of brain extract was subjected to the preclear step, and 25 ml of the supernatant was taken for IP; with 8-week-old male Sprague-Dawley rats (Charles River), 25 ml was taken for preclear step and 20 ml was taken for IP; with 12-week-old Tg2576 transgenic mice, 1.5 ml for was taken for preclear step and 1.0 ml was taken for IP; with 15-week-old male New Zealand White rabbits, 20 ml was taken for preclear step and 17.5 ml was taken for IP; and with 8-week-old male wild-type mice, 20 ml was taken for preclear step and 17.5 ml was taken for IP. H4 human neuroglioma cells (ATTC HTB-148) stably transfected with human Swedish APP were incubated for 16 h with medium at 37°C, after which the medium was collected and mixed 1:1 with 0.4% DEA/0.1 M NaCl before being stored frozen at -80°C.
MALDI-TOF MS Analysis. For each sample, the eluate containing A peptides was desalted and concentrated with a C18 ZipTip (Millipore, Billerica, MA) and then mixed with the MALDI matrix -cyano-4-hydroxycinnamic acid (Applied Biosystems, Foster City, CA). The mixture was spotted to a 192-well MALDI plate (Applied Biosystems, Foster City, CA) and analyzed using an Applied Biosystems 4700 Proteomics Analyzer, a MALDI-TOF/TOF instrument, in the linear positive ion mode. The mass range was set at 2000 to 6000 Da with the focus mass at 4300 Da. The data were collected using automatic data acquisition and a random search pattern. Each MS spectrum was an average of 10,000 laser shots with fixed laser intensity. External calibration was performed using the 4700 Cal Mix (Applied Biosystems). As appropriate for the sample, human or mouse A1–40N15 added before IP was also used as an internal standard for peak intensity and mass calibration (human peptide [M+H]+ = 4383.86; mouse peptide [M+H]+ = 4285.77). Direct mass measurements of the standard peptides with the instrument in reflectron mode confirmed these values. Identifications for tissue or cell-derived peptides were based on agreement between experimental masses and theoretical values (see Results).
To provide a basis for quantitation of changes in the relative abundance of A species, known amounts of 15N-substituted human or rat/mouse A peptides 1 to 40, 1 to 42, and 1 to 43 were spiked into each brain extract. The ratio of peak height of each peptide to the corresponding internal standard was calculated, and the changes caused by drug treatment were estimated from the change in the ratio between the peak for each tissue-derived peptide and the most relevant internal standard.
Dosing Protocol for LY-411575. Two-week-old male Hartley guinea pigs (Charles River) were dosed subcutaneously with 3.2 mg/kg LY-411575 (Wong et al., 2004) using a dosage volume of 1 ml/kg. The compound was solubilized in a vehicle of 20% dimethyl sulfoxide/20% EtOH/60% polyethylene glycol 400. Guinea pigs were euthanized 3 h after dosing, and tissues were collected. There were six animals per dosing group.
Fluorescence Polarization. The experiment was performed with a Beacon 2000 instrument, using fluorescein-A as the ligand to 6E10 antibody in phosphate buffer, pH 7.4.
Surface Plasmon Resonance. Surface plasmon resonance experiments were performed on a BIAcore 3000 instrument (BIAcore AB, Uppsala, Sweden). Biotinylated antibody was immobilized to 4000 resonance units on a BIAcore SA chip. Experiments were carried out at 25°C in buffer consisting of 20 mM Tris HCl, pH 8.5, and 50 mM NaCl. A1–40 peptide was injected over the antibody surface and an unmodified streptavidin surface at a flow rate of 10 µl/min. Binding data were referenced against the streptavidin surface and buffer injections. Dissociation data were fit to a simple exponential using BIAeval software (BIAcore AB).
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Results |
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, and 6E10, which recognizes residues 4 to 8 of human A. Conditions were sought that gave maximal depletion of the A population in a single step, with capture efficiency assessed by subjecting immunodepleted samples to a second round of IP.
In a preliminary study, either 60 µg of 4G8 (Fig. 1A) or a combination of 30 µg each of 6E10 and 4G8 (Fig. 1B) efficiently captured both endogenous A and the 15N-substituted human A1–40 internal standard from 25-ml samples of guinea pig brain extract, but 6E10 used alone (Fig. 1C) failed to capture an N-terminally truncated form of A.We elected to use a combination of the two antibodies for IP when analyzing human-like A, as this method seemed most likely at the outset of the work to yield a complete A profile.
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as a simple equilibrium for which Kd = 2 x [4G8][A]/[A-4G8], with antibody so greatly in excess over A that its free concentration [4G8] is effectively unchanged by binding all the A to antibody, an endpoint at which 
90% A is bound to antibody requires that [4G8] be 5 x Kd (because [A-4G8]/[A] = 2 x [4G8]/Kd). Kd for the interaction of 4G8 with A was reported to be 0.8 nM (Hughes et al., 1998), and the selected concentration of 4G8 at 8 nM (10-fold Kd) was therefore sufficient on its own according to theory, as well as appearing effective by experiment. Additional A binding capacity was present in the form of an equal concentration of 6E10 antibody, for which a Kd of 1.5 nM from A was measured using a fluorescence polarization assay (data not shown).
After capture, the A-antibody complexes were subjected to multiple wash steps to remove nonspecifically bound components. To evaluate the risk of losing A while washing, the off-rate for A bound to immobilized biotinyl-6E10 was measured on a BIAcore biosensor system (data not shown). The first-order rate constant for dissociation of A into the pH 8.5 wash buffer at 25°C was 3.3 x 10-4 s-1, which would result in a 10% loss in a 5-min wash. Including centrifugation, wash steps in our protocol required approximately 5 min each; however, they were performed using ice-cold buffer to retard dissociation. The available data suggested that losses by dissociation of antibody-A complexes would not have had a major effect on the IP/MS profiles obtained.
Immunoprecipitation and MALDI-MS Analysis. To check the specificity of A capture, guinea pig brain extract was subjected in turn to the IP protocol using i) bead-linked nonspecific IgG, followed by ii) bead-linked anti-A antibodies. Before the first IP, A1–40N15 was spiked into the sample as a marker for appropriately specific capture of a desired target (i.e., it should escape IP by nonspecific IgG but be captured by the anti-A antibodies). A1–42N15 was added to the captured sample from each round as a positive control for the MALDI-MS step.
No brain-derived A or A1–40N15 was captured by nonspecific IgG, because the only peak in the mass spectrum came from A1–42N15 added after IP but before MS (Fig. 2A). Subsequent IP from the same extract using A-specific antibodies produced a complex spectrum (Fig. 2B) in which the masses of 14 peaks matched theoretical values for A peptides of the guinea pig (Table 1). Assignments of peak identity were based on experimental masses that agreed within 0.05% of theory with theoretical masses for individual A species. The spectrometer was operated in linear positive ion mode to maximize its sensitivity.
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Because the guinea pig A sequence is the same as that for human, dog, and rabbit, this series of identifications applies to all four species. Table 1 also shows theoretical and experimental mass values for A of the mouse and rat and for Dutch variant human peptides, which appear later under Results.
A Peptide Profiles from Model Species. Despite the sequence-identity of guinea pig, dog, and rabbit A, there is no certainty that the -, -, and -secretase enzymes in these species will cleave APP at similar rates or with similar relative specificities for different cleavage sites. Differences in degradation or clearance of A could also cause interspecies variation in A populations. In Fig. 3, MALDI-TOF MS profiles of A are given for brain extracts of the guinea pig, dog, and rabbit (each equivalent to 2.7 g of brain tissue). The three samples gave profiles in which the principal similarities are the prominence of A species 11 to 34, 11 to 40, 1 to 33, 1 to 34, 1 to 37, 1 to 38, 1 to 40, and 1 to 42. A1–43 was also detected in all three extracts. Differences were comparatively minor and included the peak identified as A7–39 in the dog sample. The guinea pig has been recommended as an animal model for human APP/A processing because of its human-identical A, whereas the dog is favored for drug safety evaluation.
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Wild-Type and Transgenic Mice. We next examined A populations from three mouse models containing different APP substrates: wild-type mouse with its endogenous APP, as well as transgenic mice expressing either the Swedish or Dutch mutant forms of human APP (Fig. 4). Because the wild type mouse has three differences in A peptide sequence compared with human A (see Table 1), the human A-specific antibody 6E10 does not capture murine A, and so IP from the extract of wild-type mouse brain was performed with 4G8 alone. The transgenic mouse samples were probed with both 4G8 and 6E10 antibodies. Due to the different levels of total A in these mouse models, the amount of brain extract used for IP was adjusted accordingly. For mouse brain, 1.8 g was subjected to IP compared with 1.17 g of Dutch-mutant mouse brain and 0.11 g of Tg2576 mouse brain. In the Swedish mutant, a two-residue sequence change from wild-type immediately N-terminal to the BACE site enhanced the rate of A cleavage, causing an elevated ratio of A production to cleavage at the -secretase site (Citron et al., 1992). In the Dutch variant, alteration of Glu-22 to Gln-22 increases the propensity of the A population to form fibrils (Watson et al., 1999).
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pattern (Fig. 4A) distinct from that seen in guinea pig, rabbit, and dog. Its major feature was the prevalence of A peptides with N-terminal Glu-11 (Gouras et al., 1998). In contrast to the profiles shown in Fig. 3, A peptides 11 to 34, 11 to 37, 11 to 38, and 11 to 40 yielded prominent peaks, and peaks for A1–40 and A1–42 were comparatively less abundant. This was not the case for spectra from brains of transgenic mice expressing Swedish-variant (Fig. 4B) and Dutch-variant (Fig. 4C) human APP, which resembled that of wild-type guinea pig (Fig. 3A) much more than that of wild-type mouse (Fig. 4A). Both transgenic profiles were dominated by peaks of C-terminally truncated A, including A1–37, A1–38, A1–40, and A1–42, with relatively few A11–x components. The result clearly indicates how heavily minor differences in the A sequence can alter the product profile. A1–43 was not detected in any mouse models, including wild-type or human transgene-expressing models. This could be due to either a low abundance or lack of cleavage of this fragment in these models.
Transgene-derived human A dominated the profile from Tg2576 mouse (Fig. 4B), with no detection of A sourced from mouse APP. The Tg2576 model has a 6-fold higher expression of the human APP protein relative to the endogenous mouse APP, and this level of expression of the Swedish-mutation variant of APP resulted in the production of a 5-fold increase in the amount of A1–40 and a 14-fold higher amount of A1 to 42 relative to endogenous murine A (Hsiao et al., 1996). The failure to detect murine A can be accounted for by recalling that 16-fold less brain tissue was used from the Tg2576 mouse than from wild type to compensate for the robust overexpression of human A in this model. In contrast to the profile from the Tg2576 mice, endogenous mouse A peptides were clearly represented in the spectrum from the transgenic mouse expressing Dutch-variant human APP (Fig. 4C), a line that has approximately a 5-fold increase in APP expression and an approximate 4-fold excess of human A relative to murine (Fig. 4C). The quantity of brain tissue extracted for the Dutch-variant transgene was 69% of that taken for wild-type mouse. The peak profile of endogenous mouse A from Dutch-mutant mouse (Fig. 4C) was consistent with the profile from wild-type mouse (Fig. 3A), with prominent A11–x peaks (mouse A11–34, A11–38, and A11–40). The striking feature of the results from Dutchvariant mouse is coexistent within a single brain of different processing patterns for the mouse and human APPs. On a technical point, the murine A profile obtained from mice carrying the Dutch variant APP transgene by IP using both monoclonals generally matched the profile captured from wild-type mice by 4G8 alone. This mitigates any concern that use of 4G8 alone might skew the profile in favor of A11–x components.
A Secreted from Human Cells. Stably transfected cell lines that produce A are also used in AD research. Figure 5 shows MALDI-TOF MS profiles for A secreted from H4 human neuroglioma cells stably transfected with either wild-type human APP (Fig. 5A) or Swedish-variant APP (Fig. 5B). In each case, A was immunoprecipitated from cell-conditioned medium with 6E10 and 4G8 antibodies. A notable difference between the profiles is the relative loss of A11–x peptides in the H4Sw/APP-conditioned medium (Fig. 5B), which can be attributed to the previously detected influence of the Swedish mutation on the relative rates of cleavage before Asp-1 and Glu-11 of A. The spectra in Fig. 5, together with those from the Tg2576 mouse brain (Fig. 4B), support the view that the Swedish mutation favors production of A1–x relative to other potential events (e.g., -secretase-catalyzed cleavage or production of A11–x) (Citron et al., 1994) and also imply that BACE may cleave before either Asp-1 or Glu-11 but not function sequentially at both sites.
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production. These include the guinea pig (Fig. 6A), which produces human-identical A peptides, the wild-type mouse (Fig. 6B), and the wild-type rat (Fig. 6C). Mouse and rat generate almost identical A products that differ from the human sequence at three positions. The result of the comparison is clear. The species that produce human-identical A species give profiles in which A1–34 and A1–40 are the largest peaks, whereas the mouse and rat profiles are dominated by A peptides beginning at Glu-11 of the A1–40 sequence.
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Peptide Production in Guinea Pig Brain. The ability to detect and identify individual A peptides in a complex pool should allow the evaluation of drug effects on individual peptides in model species. Drug action is quantified by noting how peaks from tissue-derived A change in comparison with peaks for internal standards, which were added at constant levels to all brain extracts. Multiple internal standards allowed more precise quantitation of specific A peptides. The initial selection of internal standards A1–40N15 and A1–42N15 to calibrate the corresponding endogenous A1–40 and A1–42 in guinea pig brain extracts was based on their known importance and disease relevance and the greater variability that might be expected for longer and more hydrophobic A products. The A1–40N15 peptide also served as internal standard for shorter A peptides, because it was not feasible to have an independent standard for every A in the pool.
Figure 7 shows the effect of a 3.2 mg/kg dose of a -secretase inhibitor LY-411575 (Eli Lilly & Co., Indianapolis, IN), on the A profile from guinea pig brain (see Materials and Methods for details). LY-411575 generally inhibited production of A in guinea pig brain, with most fragments declining as gauged by their peak intensity relative to the internal standards. An exception was the elevation of A1–43, which appeared to be an increase of severalfold by reference to the both the A-40N15 and A1–42N15 internal standards. Although a corresponding A1–43 internal standard was not included in this experiment, the robust increase in endogenous A1–43 is readily quantified with the two internal standards present, and subsequent experiments prove that employing all three internal standards would not change the outcome of this experiment. Effects of this kind would be difficult to detect without the capacity of mass spectrometry to survey the A population.
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Discussion |
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in human brain (Huse et al., 2002), in cerebrospinal fluid of AD patients (Lewczuk et al., 2003; Portelius et al., 2006), or in conditioned media from cells transfected with genes encoding human APP (Wang et al., 1996; Vandermeeren et al., 2001). There have been fewer studies of A populations in animal brains (Terai et al., 2001; Lewis et al., 2004; Rufenacht et al., 2005), despite the potential value of such data in interpreting the properties of important models and the actions of drug candidates.
Because different A peptides contribute differently to neurotoxic insult and plaque formation (Parvathy et al., 2001), understanding interspecies differences in A peptide production should help to rationalize differences among different in vivo models. In addition, the enzymology of -secretase includes subtle responses to pharmacologic intervention, such as uneven shifts in the relative quantities of particular A peptides (Ikeuchi et al., 2003; Beher et al., 2004). As a profiling method, IP/MS offers an excellent approach to both these subjects.
The most favored animal models are transgenic mice that overexpress mutant or wild-type human APP and exhibit amyloid deposits. They express both human and mouse APP at different ratios in the presence of either endogenous murine PS1 [the Tg2576 mouse (Hsiao et al., 1996)] or transgenic human PS1 [the PSAPP mouse (Takeuchi et al., 2000)]. The three differences in sequence between human and mouse A cause profound differences among their respective biophysical properties and propensities to form oligomers and plaques (De Strooper et al., 1995). IP/MS was used here to analyze whether A profiles from transgenic mice expressing different forms of human APP had more resemblance to those from animals that make human-identical A or to those from animals that make wild-type rat/mouse A.
The low level of A in brains of animals lacking an APP transgene provided a technical challenge. Our method was designed to capture and detect even minor A species from relatively large amounts of brain extract for the purpose of thorough characterization of A in animal brains, including both extracellular and intracellular forms. It clearly detected the fragments already implicated in AD, as well as recognizing several A fragments not reported elsewhere (to our knowledge). As a critical step, components that bound nonspecifically to IgG were removed before specific A capture, minimizing interference with capture and mass analysis.
A profiles from brain extracts of guinea pig, dog, and rabbit, three species that make human-identical A (Johnstone et al., 1991), were similar (Fig. 3) except for a small peak of A7–39 from dog brain. The longer A forms identified were A1–40, A1–42, and A1–43. Another major peak was A1–34; cleavage of -Leu34-Met35- in A is attributed to BACE (Shi et al., 2003). The peptide profile from untreated guinea pig was consistent across all experiments (Figs. 3, 6, and 7), demonstrating the reproducibility of the pattern of A peptides obtained with this methodology.
To gauge how different APP sequences affected A profiles, we next analyzed brain A from wild-type mice and two transgenic lines expressing mutant human APP (Fig. 4). The transgenics were i) Tg2576 mice, expressing Swedish-variant human APP, and ii) mice expressing Dutch-variant human APP (in which Glu-22 of A becomes Gln-22). All three lines produce A by activity of endogenous mouse secretases. IP/MS of wild-type mouse brain showed the preponderance of A11–x species reported earlier (Gouras et al., 1998); cleavage of -Phe10-Glu11- in mouse/rat A is attributed to BACE1 (Vassar et al., 1999). Each transgenic model produced a distinctive profile. That from Tg2576 brain was dominated by human-sequence A and resembled the guinea pig, dog, and rabbit profiles. Dutch-variant APP mouse gave a complex profile containing human and murine As, but peaks from each APP were easily assigned to their respective A sequences. The A profile derived from murine APP matched the one from wild-type mouse, and the A profile from the Dutch-type human APP was generally similar to the one from animals containing human-identical A.
Comparing profiles from the three mouse models again suggested that it was the APP substrate (presumably its sequence in the A and pre-A regions) rather than host secretase and A-degrading enzymes that determined the A profile. Further support came from comparing the native A profiles of three different rodent species (Fig. 6). The similar A profiles of mouse (Fig. 6B) and rat (Fig. 6C) were dominated by peaks of rat/mouse A11–34 and A11–40 and were distinct from the guinea pig profile (Fig. 6A), with major peaks of A1–40 and A1–34.
The A population secreted from H4 cells expressing wild-type human APP included a strong peak of A11–40 (Fig. 5A); however, A11–40 was barely detected in medium conditioned by cells expressing Swedish variant (Fig. 5B). This presumably reflected the enhanced kinetics of cleavage at the A N terminus with Swedish-variant APP as substrate. Tg2576 mice, which express the Swedish human variant, as well as H4 cells overexpressing Swedish-variant APP, produced a profile dominated by A1–40, with almost no A cleaved after Tyr-10. This was consistent with the report that skin fibroblasts from persons of the Swedish pedigree secreted elevated levels of A, with peptides starting at A1 predominant (Citron et al., 1994).
BACE and -secretase are drug targets in AD, and IP/MS profiling could apply to either approach. Surprisingly, the more complex -secretase has yielded more quickly to medicinal chemistry in terms of getting trial agents to the clinic. To us and others (Beck et al., 2003), the guinea pig has attractions as a preclinical model that include its human-like brain A profile (e.g., compare Figs. 5A and 6A). This made it interesting to test the effects of a -secretase inhibitor on the A profile from guinea pig brain.
IP/MS is unique in distinguishing so many different A fragments, and the use of 15N-substituted A standards allows quantitative assessment of drug effects on all brain A peptides simultaneously. A -secretase inhibitor, LY-411575, was shown to have induced acute changes in the guinea pig brain A profile 3 h after the animal received a subcutaneous dose of 3.2 mg/kg (Fig. 7). A1–40 and A1–42 were both diminished in abundance relative to their specific internal standards. The other A fragments were also diminished, including A1–34, a product of two BACE-catalyzed cleavages. This phenomenon was interpreted elsewhere as signifying that -secretase must cleave the CT99 fragment of APP before BACE cleaves after Leu-34 (Shi et al., 2003). It was also observed that the -secretase inhibitor diminished some fragments more than others, which adds further complexity to the process of secretase inhibition. Of particular interest, the A1–43 fragment was increased more than 2-fold over vehicle levels in drug-dosed animals. We have replicated this result in a second experiment (data not shown). Similar results were observed with cellular systems (Ikeuchi et al., 2003; Qi-Takahara et al., 2005) but have not (to our knowledge) been reported before in animal brain. The importance of A1–43 is not fully understood, but its strongly amyloidogenic nature presumably makes it undesirable (Burdick et al., 1992; Jarrett et al., 1993). Consistent with this, Ax–42 and Ax--43 were shown to be associated with early AD progression in human patients (Parvathy et al., 2001). This result underlines the complexity of the task of pharmacologically inhibiting A processing through secretase inhibition, reinforcing the importance of thoroughly studying the A profile in animal models. As we detected A1–43 only in animal models endogenously expressing the human A sequence (and flanking sequence), the proper selection of animal models and detailed A profiling may promote understanding the effects of secretase inhibitors and their potential impact in human brain.
Judging by the A profiles presented here for in vivo models and in vitro models, the APP sequence is the principal factor governing the A profile. According to the amyloid hypothesis, the production and toxic effects of A peptides are the primary cause of neurotoxicity and consequent cell death in the brain. The impossibility of direct biochemical analyses on brain tissue from living patients makes it immensely important that animal models of the disease be chosen based on maximal information. A thorough understanding of species differences in A peptide production will enhance our understanding of in vivo models and thereby promote efforts to defeat AD. The IP/MS described above revealed the complexity of APP processing in different species. The unexpected observation of enhanced production of A1–43 caused by a -secretase inhibitor further emphasized the importance of thoroughly assessing the response to drug treatment at a high level of detail. That this result is obtained in an animal model that closely resembles A processing in the normal human brain underlines the importance of attempting to gauge the medical potential of emerging pharmaceutical A modifiers in a human-like biological context.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: A, -amyloid; AD, Alzheimer's disease; APP, amyloid precursor protein; BACE, -amyloid-converting enzyme or -secretase; DEA, diethylamine; MALDI-TOF MS, matrix-assisted laser desorption time-of-flight mass spectrometry; IP, immunoprecipitation; IP/MS, immunoprecipitation followed by mass spectrometry; Tg, transgenic; A1-40N15, 15N-substituted A1–40; PS, presenilin; PS1, presenilin-1; PS2, presenilin-2; LY411575, N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[bid]azepin-7yl]-L-alaninamide.
Address correspondence to: Barbara Tate, Pfizer, Inc., Eastern Point Rd., MS# 8220-4070, Groton, CT 06340. E-mail: barbara.tate{at}pfizer.com
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