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. 2009 Mar;174(3):722–726. doi: 10.2353/ajpath.2009.080790

E22Q-Mutant Aβ Peptide (AβDutch) Increases Vascular but Reduces Parenchymal Aβ Deposition

Martin C Herzig *, Yvonne S Eisele *†, Matthias Staufenbiel ‡, Mathias Jucker *
PMCID: PMC2665734  PMID: 19218342

Abstract

Patients that have hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) generate both wild-type β-amyloid (Aβwt) and E22Q-mutant β-amyloid (AβDutch). Postmortem analysis of HCHWA-D brains reveals severe cerebral amyloid angiopathy with very little parenchymal amyloid deposition. To investigate amyloidosis in the presence of both Aβwt and AβDutch variants, transgenic (tg) APP23 mice were crossed with APPDutch mice. Although single-tg APP23 mice deposited Aβwt with aging, double-tg APP23/APPDutch mice co-deposited AβDutch (mainly AβDutch1-40) and Aβwt at twofold higher total Aβ levels. Vascular Aβ deposits and hemorrhages were twice as high in APP23/APPDutch mice compared with APP23 mice. Surprisingly, parenchymal Aβ deposition was reduced in the double-tg mice compared with the single-tg APP23 mice. Our findings suggest that AβDutch1-40 inhibits parenchymal amyloidosis but exacerbates vascular amyloid, hence explaining the compartment-specific distribution of cerebral amyloid in HCHWA-D patients.

Patients carrying familial mutations within the β-amyloid (Aβ) sequence of the amyloid precursor protein (APP) develop cerebral amyloid angiopathy (CAA) with or without additional parenchymal amyloid plaques.1,2 Because such patients are heterozygous for the mutation, they produce both wild-type (wt) and mutant Aβ. Several in vitro studies have demonstrated that mutated Aβ mixed with Aβwt shows altered aggregation and toxicity compared with Aβwt or mutated Aβ alone.1 Nevertheless current transgenic (tg) mice that model familial cerebral amyloidosis caused by an APP mutation within the Aβ sequence express only the mutant human Aβ but lack the complementary human Aβwt counterpart.

Patients affected by hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) develop severe CAA and exhibit fatal hemorrhagic strokes typically in their 50s.3 These patients carry an E693Q APP mutation corresponding to E22Q of Aβ. To study an in vivo model that mimics HCHWA-D, we have crossbred APPDutch tg mice that express E22Q Aβ (AβDutch)4 with homozygous APP23 mice expressing Aβwt.5 Double-tg APP23/APPDutch mice were aged to 24 months and compared with single-tg APP23 littermates.

Materials and Methods

Transgenic Mice

The generation of APPDutch and APP23 tg mice has been described previously.4,5 APPDutch mice overexpress E693Q-mutated human APP751 whereas APP23 mice overexpress KM670/671NL-mutated APP751 (Swedish mutation). Both lines express their transgenes under the murine Thy-1 promoter element and have either been generated on a C57BL/6J background (APPDutch) or backcrossed to C57BL/6J mice for more than 10 generations (APP23). Homozygous APP23 mice were crossbred with hemizygous APPDutch mice. To distinguish double-tg APP23/APPDutch mice from single-tg APP23 mice levels of human APP and Aβ were assessed by Western blotting. A total of 23 mice was used: four female APP23/APPDutch versus eight female APP23 mice; and four male APP23/APPDutch versus seven male APP23. All animals were 24 months of age. The experiments were in compliance with protocols approved by the local animal care and use committees.

Human Tissue

HCHWA-D tissue was from a 76-year-old male patient (provided by M.L.C. Maat-Schieman, Leiden University Medical Center, Leiden, The Netherlands). Tissue from a 61-year-old female with Alzheimer’s disease (AD) was generously provided by M. Tolnay University of Basel (Basel, Switzerland).

Immunohistochemistry

Mouse brains were carefully removed while it was made sure that the leptomeningeal vessel remained attached to the brain. One hemisphere was immersion-fixed in 4% paraformaldehyde (the other half of the brain was used for Western blotting). Immunohistochemistry for Aβ was done on 25-μm-thick, free-floating sagittal sections with polyclonal antibody NT12 (similar to NT11),5 using standard immunoperoxidase procedures (Vector Laboratories, Burlingame, CA). NT12 antibody has been shown to recognize both wild-type and Dutch-mutated Aβ.4

Quantification of Total Aβ Load and CAA

The Aβ load was quantified on Aβ-immunostained sets of every 12th systematically sampled serial sagittal 25-μm-thick section (typically yielding 12 sections/hemibrain). Images of sections were acquired with a scanner (Perfection 3200 Photo; Epson, Long Beach, CA) using transmitted light and processed with Adobe Photoshop CS (Adobe Systems, San Jose, CA) and the area of the forebrain (reported in percent) covered by Aβ-immunostaining was determined with the Analyze Particles tool of ImageJ (National Institutes of Health, Bethesda, MA; http://rsb.info.nih.gov/ij/). Thus, Aβ load was defined as total parenchymal and vascular Aβ-immunostained profiles. Quantitative analysis of CAA was performed on the same NT12-immunostained sections. The number of Aβ-positive vessels throughout the neocortex and the overlying leptomeningeal vessels was counted manually using an Axioskop 2 microscope with a ×10 objective (Zeiss, Oberkochen, Germany). A grading system dividing Aβ-positive vessels in one of three severity grades was applied as previously described.6 CAA grade 1: Aβ-immunoreactivity is restricted to the vessel wall; CAA grade 2: Aβ-immunoreactivity extends focally to the surrounding neuropil; CAA grade 3: extensive infiltration of Aβ into the parenchyma with a complete amyloid coat around the vessel. Neighboring profiles of immunoreactive vessel were considered as separate vessels.

Quantification of Cerebral Hemorrhage

Perls’ Berlin Blue-stained clusters of hemosiderin-positive microglia were quantified on an additional set of every 12th systematically sampled sections6 throughout the neocortex and the overlying leptomeningeal vessels.

Western Blot Analysis

For analysis of total human Aβ and APP, we performed multiphasic urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. This gel system allows to separate Aβ1-40 from Aβ1-42 and Aβwt from AβDutch as described previously.4,7,9 Briefly, homogenized mouse brain hemispheres (forebrain) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% T, 5% C bicine/Tris minigels containing 8 mol/L urea in the separation gel. Proteins were transferred to a polyvinylidene difluoride Immobilon-P membrane by semidry blotting, incubated with antibody 6E10 (Covance, Princeton, NJ) and visualized by chemiluminescence (ECL; Amersham, Arlington Heights, IL). Antibody 6E10 recognizes residues 1 to 16 of Aβ, and the Dutch mutation at position 22 does not interfere with its binding. Synthetic AβDutch was a gift of J. Ghiso (New York University School of Medicine, New York, NY). Intensities of both human Aβ and APP bands (4 μl of 1:44 diluted samples were loaded) were quantified with the Gel Analyzer Tool of ImageJ (http://rsb.info.nih.gov/ij/).

Statistical Analysis

Unpaired t-test was used for group comparisons using Microsoft Excel software (Microsoft Corporation, Redmond, WA). Values are reported as means ± SEM. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

Results

Western blotting revealed that human APP levels in aged double-tg APP23/APPDutch mouse brain were at least twice those observed in single-tg APP23 mice (Figure 1A). While cerebral amyloid in APP23 mice was composed of both Aβwt40 and Aβwt42 with Aβwt40 exceeding Aβwt42 (Figure 1A), APP23/APPDutch mice exhibited an additional Aβ40Dutch band in its intensity similar to Aβwt40. The levels of AβDutch42 remained below the limit of detection, similar to those previously reported for single-tg APPDutch mice.4 Consistently, quantitative analysis of total Aβ40 (ie, wild-type and mutated Aβ combined) revealed an approximately twofold significant increase in APP23/APPDutch mice compared with APP23 mice, with no difference in total Aβ42 (Figure 1, B and C).

Figure 1.

Figure 1

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APP and Aβ levels in brains of APP23/APPDutch and APP23 mice. A: Western blot analysis of human APP and Aβ in 24-month-old mouse forebrains (n = 23; females: four APP23/APPDutch and eight APP23 mice; males: four APP23/APPDutch and seven APP23). Top: APP levels in double-tg APP23/APPDutch mice (lanes 4 and 6) were at least twice as high compared with those in littermate single-tg APP23 mice (lanes 5 and 7). Bottom: Synthetic human Aβwt1-40 and Aβwt1-42 (lane 1), AβDutch1-40 (lane 2), and AβDutch1-42 (lane 3) peptides were used as markers. Whereas cerebral amyloid of single-tg APP23 mice was mainly composed of Aβwt40 and with lower levels of Aβwt42 (lanes 5 and 7), double-tg APP23/APPDutch mice showed an additional AβDutch40 band (lanes 4 and 6) with an intensity similar to that of Aβwt40. AβDutch42 remained below detection level (lanes 4 and 6). The Aβ composition of an HCHWA-D brain (lane 8) was similar to that of APP23/APPDutch brains (lanes 4 and 6) with the exception that Aβwt42 was readily detectable in the double-tg mouse brains. Western blot analysis of Aβ in AD brain (lane 9) resembled that of single-tg APP23 brains (lanes 5 and 7). B and C: Densitometric analysis of Aβ levels in the female mice revealed significant approximately twofold higher total Aβ40 levels (composed of Aβwt and AβDutch) in double-tg APP23/APPDutch versus single-tg APP23 littermates (unpaired Student’s t-test; ***P < 0.001) whereas Aβ42 levels were similar in the two genotypes (P > 0.05). In male mice (results not shown) the increase in total Aβ40 levels in APP23/APPDutch was 1.6-fold (P < 0.01) with again no change in Aβ42 levels compared with APP23 littermates.

Aβ immunohistochemistry of both APP23/APPDutch and APP23 brains revealed parenchymal and vascular Aβ deposition. Surprisingly however, total cerebral Aβ load in APP23/APPDutch mice was lower than that in APP23 littermates (Figure 2, A–C) despite the overall higher levels of total Aβ (Figure 1). In contrast, quantification of the number of Aβ-positive neocortical vessels (including the overlying leptomeningeal vessels), revealed a nearly twofold increase in APP23/APPDutch mice compared with APP23 mice (Figure 2D). We have previously shown that APPDutch mice at a similar age (22 to 24 months) develop only low levels of Aβ deposition, which is restricted to vessels.4,8 Previous quantification revealed only approximately nine Aβ-positive neocortical vessels (including the overlying leptomeningeal vessels) per section,8 which is only a quarter of the Aβ-immunopositive vessels in APP23 mice described in the present study. Vascular amyloid is a known cause of intracerebral hemorrhage in humans and APP tg mice2,6 and thus neocortical and leptomeningeal hemorrhages were additionally analyzed. Consistent with the increase in CAA, results revealed an approximately twofold increase in microbleedings in APP23/APPDutch mice compared with APP23 mice (Figure 2E).

Figure 2.

Figure 2

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Cerebral amyloidosis and hemorrhage in APP23/APPDutch and APP23 mice. The same mice as indicated in Figure 1 were used. A–C: Immunohistochemistry for Aβ revealed a significantly higher total Aβ load in the forebrains of female single-tg APP23 mice compared with female double-tg APP23/APPDutch mice (n = 8 and 4, respectively; unpaired Student’s t-test; **P < 0.01) despite the observation that total Aβ levels were higher in double-tg compared with the single-tg mice (see Figure 1). The same was true for the male mice (n = 7 and 4, respectively; P < 0.05; not shown). D: Vascular Aβ load, however, was significantly higher in the female double-tg APP23/APPDutch mice compared with the single-tg APP23 mice, and this was true for total neocortical and leptomeningeal vessels (**P < 0.01) as well as for individual cortical grade assessment C1 to C3 (P < 0.05). A similar increase in vascular Aβ load was found for the males (not shown). E: Quantification of neocortical and leptomeningeal hemorrhages revealed a significant, approximately twofold, increase in hemorrhages in female APP23/APPDutch mice versus APP23 mice (*P < 0.05). Again, a similar increase was also found for the males (not shown). C, cortical; C1 to C3, cortical grade 1 to 3; L, leptomeningeal. Scale bar = 500 μm.

Discussion

Cerebral amyloid in HCHWA-D patients consists of both AβDutch and Aβwt, with the vast majority being of the Aβ40 species (present results and 4, 9, 10). In contrast, patients with AD reveal both Aβ40 and Aβ42 with Aβ42 being the disease-driving and more toxic species.11 Several studies have indicated that the ratio of Aβ42 to Aβ40 determines in which compartment cerebral amyloid deposition occurs, i.e., a high Aβ42/Aβ40 ratio promotes parenchymal amyloid deposition whereas a low ratio promotes CAA, and this appears to hold true for both Aβwt and AβDutch.1 The reason why the Aβ42/Aβ40 ratio determines the cerebral compartment in which amyloid deposition occurs is not entirely clear. At least in transgenic mice using the Thy-1 promoter, cerebral Aβ is neuron-derived and must subsequently be transported to the vasculature (possibly as part of its drainage along the periarterial fluid pathway or into the blood for clearance).12,13 Aβ42 aggregates more readily than Aβ40 and thus, it is more likely to form an amyloid seed during its transport from the neuron to the vasculature.

The present finding that double-tg APP23/APPDutch mice develop fewer parenchymal Aβ deposits and significantly more CAA compared with single-tg APP23 mice reveals that the observed amyloid pathology in APP23/APPDutch mice is not a simple addition of the amyloid patterns seen in the corresponding single-tg mice. In particular, the reduced parenchymal Aβ deposition in APP23/APPDutch mice was unexpected in light of the higher levels of total Aβ in double-tg compared with single-tg mice. In addition, the twofold increase in vascular Aβ-deposits observed in APP23/APPDutch versus APP23 brains cannot the explained by a simple additive effect because 24-month-old single-tg APPDutch mice reveal only few Aβ deposits that are restricted to the vessels. Quantitatively, CAA in 24-month-old APPDutch mice is only a quarter of the amount found in single-tg APP23 mice.4,8 Although the (lower) Aβ42/Aβ40 ratio in the APPDutch/APP23 mice compared with the APP23 mice may be used to explain the increased CAA as well as the reduced parenchymal Aβ deposition, this explanation falls short if compared with single APPDutch mice, because APPDutch mice generate approximately eightfold more Aβ40 compared with Aβ42.4 Thus, on one hand the total Aβ generation seems to be an important variable. On the other hand, in familial AD, the Aβ42/Aβ40 ratio appears more important than the total Aβ to drive the deposition because some of the familial AD mutations in the presenilins specifically decrease Aβ40 rather than increasing Aβ42.14 From this and other observations,15 Aβ40 was suggested to be inhibitory for parenchymal amyloid deposition. Indeed, in vitro Aβ40 retards the aggregation of Aβ42 in a concentration-dependent manner.16,17

Introducing the E22Q substitution to Aβ results in a less efficient clearance of the mutated peptide from the brain, both by enzymatic cleavage and receptor-mediated transport into CSF/blood.1 AβDutch also shows a higher affinity to smooth muscle cells, where the initiation of vascular amyloid formation was suggested to occur.18 In vitro, AβDutch reveals higher oligomerization and aggregation propensity compared with Aβwt.19,20,21 Most relevant for the present study and the pathomechanism of HCHWA-D is the observation that mixing Aβwt with AβDutch alters and enhances aggregation propensities of either wild-type or mutated Aβ alone.1,22 Thus, it is likely that heterogeneous aggregates, protofibrils, or fibrils made of both AβDutch and Aβwt have different biological propensities compared with homogeneous Aβ species made of either wild-type or mutated Aβ alone. The mouse models used in the present study also express a small amount of murine Aβ and thus, it cannot be ruled out that murine Aβ additionally influences the biological properties of the human Aβ species.

In conclusion, our results suggest that the expression of E22Q-mutated Aβ (primarily AβDutch40) is inhibitory for parenchymal amyloidosis but exacerbates vascular amyloidosis, therewith explaining the compartment-specific distribution of cerebral amyloid observed in HCHWA-D patients. The finding that the amyloid lesions in APP23/APPDutch mice are not a simple overlay of the amyloid lesions in the corresponding single-tg mice suggests that Aβwt and AβDutch interact in mice as well as in HCHWA-D patients.

Acknowledgments

We thank Claudia Schäfer, Jörg Odenthal, Simone Eberle (Tübingen, Germany), and Dorothee Abramowski (Basel, Switzerland) for experimental help; Jorge Ghiso (New York, NY) for the synthetic AβDutch peptides; and Markus Tolnay (Basel, Switzerland) and Marion Maat-Schieman (Leiden, The Netherlands) for the human brain tissue.

Footnotes

Address reprint requests to Mathias Jucker, Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Otfried-Müller Strasse 27, D-72076 Tübingen, Germany.E-mail: mathias.jucker@uni-tuebingen.de.

Supported by the German National Genome Network (grants NGFN2 and NGFNPlus).

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