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The Journal of Neuroscience, January 17, 2007, 27(3):627-633; doi:10.1523/JNEUROSCI.4849-06.2007

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Neurobiology of Disease
Aß40 Inhibits Amyloid Deposition In Vivo

Jungsu Kim, Luisa Onstead, Suzanne Randle, Robert Price, Lisa Smithson, Craig Zwizinski, Dennis W. Dickson, Todd Golde, and Eileen McGowan

Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, Florida 32224

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Numerous studies have established a pivotal role for Aß42 in Alzheimer's disease (AD) pathogenesis. In contrast, although Aß40 is the predominant form of amyloid ß (Aß) produced and accumulates to a variable degree in the human AD brain, its role in AD pathogenesis has not been established. It has generally been assumed that an increase in Aß40 would accelerate amyloid plaque formation in vivo. We have crossed BRI-Aß40 mice that selectively express high levels of Aß40 with both Tg2576 (APPswe, K670N+M671L) mice and BRI-Aß42A mice expressing Aß42 selectively and analyzed parenchymal and cerebrovascular Aß deposition in the bitransgenic mice compared with their singly transgenic littermates. In the bitransgenic mice, the increased steady-state levels of Aß40 decreased Aß deposition by 60–90%. These results demonstrate that Aß42 and Aß40 have opposing effects on amyloid deposition: Aß42 promotes amyloid deposition but Aß40 inhibits it. In addition, increasing Aß40 levels protected BRI-Aß40/Tg2576 mice from the premature-death phenotype observed in Tg2576 mice. The protective properties of Aß40 with respect to amyloid deposition suggest that strategies that preferentially target Aß40 may actually worsen the disease course and that selective increases in Aß40 levels may actually reduce the risk for development of AD.

Key words: Alzheimer's disease; amyloid ß; aggregation; premature death; transgenic mice; cerebral amyloid angiopathy

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Accumulation of amyloid ß (Aß) is hypothesized to initiate a pathogenic cascade that eventually results in Alzheimer's disease (AD) (Hardy and Selkoe, 2002). Sequential amyloid ß precursor protein (APP) processing by ß-secretase and -secretase produces a major Aß species, Aß1-40, and a number of minor species, including Aß1-42 (Steiner and Haass, 2000). Studies of AD-causing mutations in APP, presenilin 1 (PSEN1), and presenilin 2 (PSEN2) genes demonstrate that the vast majority of these mutations alter APP processing in a manner that either increases the absolute or relative levels of Aß42 (Price et al., 1998). In vitro, Aß42 aggregates into amyloid much more rapidly than Aß40 (Caughey and Lansbury, 2003). In vivo, Aß42 is the predominant form of Aß that accumulates in the AD brain and is essential for seeding Aß deposition (Younkin, 1998; Fryer and Holtzman, 2005).

Previously, we have described transgenic mice that selectively express either Aß1-40 or Aß1-42 in the secretory pathway without human APP overexpression by fusing Aß40 or Aß42 peptide sequences to the C-terminal end of the BRI protein (McGowan et al., 2005). BRI-Aß40 mice expressing high levels of Aß40 had no pathology at any age. In contrast, BRI-Aß42A mice expressing 10-fold lower levels of Aß42 developed amyloid deposits in the cerebellum as early as 3 months (McGowan et al., 2005). These data suggest that Aß42, but not Aß40, is sufficient to drive amyloid deposition in vivo. Such studies demonstrate a key role for Aß42 in initiating AD pathology but do not provide a great deal of insight into the role that Aß40 plays in AD pathogenesis.

Aß40 does accumulate in the AD brain, but the extent of Aß40 accumulation relative to Aß42 is highly variable and is usually attributed to accumulation of Aß40 in cerebral vessels (Gravina et al., 1995). Given that Aß40 is the predominant form of Aß produced and that therapeutic strategies targeting Aß typically do not selectively target any single Aß species, we have conducted experiments to directly examine the contribution of Aß40 to amyloid deposition in vivo. We bred BRI-Aß40 mice with both Tg2576 mice and BRI-Aß42A mice. The bitransgenic mice from crossbreedings had increased steady-state soluble Aß levels but significantly less parenchymal and vascular amyloid deposition compared with their respective single transgenic Tg2576 or BRI-Aß42A littermates. These results demonstrate Aß40 has anti-amyloidogenic effect in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of mice. Transgenic mice expressing BRI-Aß40 and BRI-Aß42 under the control of mouse prion promoter were generated as described previously (McGowan et al., 2005). Hemizygous BRI-Aß40 mice or BRI-Aß42A mice were crossed with hemizygous Tg2576 (APP_swe) mice (Hsiao et al., 1996). To generate the bitransgenic BRI-Aß40/BRI-Aß42 mice, hemizygous BRI-Aß40 mice were mated with hemizygous BRI-Aß42A or BRI-Aß42B mice. BRI-Aß mice were maintained on a B6/C3 hybrid background, and Tg2576 mice were maintained on a B6/SJL background. All animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee.

Quantification of parenchymal amyloid deposition. Hemibrains were immersion fixed in 10% formalin and processed for paraffin embedding. Brain tissue sections (5 µm) were immunostained with anti-total Aß antibody (Ab) (33.1.1, 1:1000; a gift from T. Golde, Mayo Clinic) on a Dako (Glostrup, Denmark) autostainer. Sections were counterstained with hematoxylin. Six sections per brain through the hippocampus, piriform cortex (bregma, –1.70 to –2.80 mm), or cerebellum (paraflocculus, crus ansiform, and simple lobules; bregma, –5.40 to –6.36 mm) were used for quantification (n = 5–7 mice per genotype at each age group). The Aß plaque burden was determined using MetaMorph software (Molecular Devices, Palo Alto, CA). For quantification of cored plaques, serial sections of those analyzed for Aß burden were stained with thioflavine S (ThioS), and the number of ThioS-positive plaques in the hippocampus, entorhinal/piriform cortex, or the cerebellum was counted. All of the above analyses were performed in a blinded manner.

Quantification of vascular amyloid deposition. For quantification of cerebral amyloid angiopathy (CAA), 5 µm paraffin-embedded sections at 30 µm intervals through the parietal or cerebellar cortex leptomeninges were immunostained with biotinylated-Ab9 antibody (anti-Aß1-16, 1:500; a gift from T. Golde) overnight at 4°C (n = 5–7 mice per genotype at each age group, n = 6 sections per mouse). Positively stained blood vessels were visually assessed using modified Vonsattel's scoring system as described previously (Greenberg and Vonsattel, 1997). The CAA severity score was calculated by multiplying the number of CAA vessels with the CAA severity grade.

Aß sandwich ELISA. For brain Aß ELISAs, forebrain and hindbrain Aß levels were determined independently, and the olfactory bulb was excluded from analysis. For plasma Aß analysis, blood was collected in EDTA-coated tubes after cardiac puncture. Blood samples were centrifuged at 3000 rpm for 10 min at 4°C, and the plasma was aliquoted and stored at –80°C until used. Aß levels were determined by end-specific sandwich ELISAs using Ab9 (anti-Aß1-16 Ab) as the capture Ab for Aß40, 13.1.1–HRP (anti-Aß35-40 Ab) as the detection Ab for Aß40, 2.1.3 (anti-Aß35-42 Ab) as the capture Ab for Aß42, and Ab9–HRP as the detection Ab for Aß42, as described previously (Kawarabayashi et al., 2001) (n = 5–7 mice per genotype at each age group). Aß levels were normalized to our previous results using the same sets of mice as internal controls to minimize potential ELISA variability.

Survival analysis. Survival rates were analyzed using Kaplan–Meier methods. Holm–Sidak methods (post hoc) were used for all pairwise multiple comparison tests (SigmaStat 3.0; Systat Software, San Jose, CA). The extraneous deaths were censored. All comparisons were made between littermates to limit any potentially confounding effects from background strain differences.

Western blotting. Snap-frozen forebrain samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Boston BioProducts, Worcester, MA) with 1% protease inhibitor mixture (Roche, Indianapolis, IN). The homogenate was centrifuged at 100,000 x g for 1 h at 4°C. Protein concentration in supernatants was determined using the BCA protein assay (Pierce, Woburn, MA). Protein samples (20 µg) were run on Bis-Tris 12% XT gels or Bis-Tris 4–12% XT gels (Bio-Rad, Hercules, CA) and transferred to 0.2 µm nitrocellose membranes. Blots were microwaved for 2 min in 0.1 M PBS twice and probed with Ab 82E1 (anti-Aß1-16, 1:1000; IBL, Gunma, Japan) and CT20 (anti-APP C-terminal 20 amino acids, 1:1000; a gift from T. Golde). Blots were stripped and reprobed with anti ß-actin (1:1000; Sigma, St. Louis, MO) as a loading control. Relative band intensity was measured using ImageJ software.

In vitro Aß aggregation assay. Synthetic Aß40 or Aß42 peptides (Bachem, Torrance, CA) were dissolved in DMSO and diluted in TBS at various molar ratios as indicated. Aß mixtures were either directly used for analysis or incubated for 2 h at 37°C without shaking. Mixtures were run on 4–20% Tris-HCl gels under nondenaturing conditions and transferred to a 0.4 µm polyvinylidene difluoride membrane as described previously (Klug et al., 2003). The blot was probed with 1:1000 Ab9.

Statistical analysis. Aß levels, amyloid plaque burden, and CAA severity were analyzed by using ANOVA with the post hoc Holm–Sidak multiple comparison test or two-tailed Student's t test (SigmaStat 3.0). If the data set did not meet the parametric test assumptions, either the Kruskal–Wallis test followed by the post hoc Dunn's multiple comparison or the Mann–Whitney rank sum test was performed (SigmaStat 3.0). To test whether the Aß levels in the bitransgenic mice were consistent with an additive sum of Aß levels in the single transgenic littermates, a multiple linear regression with no intercept test was used (StatsDirect 2.5.6). All comparisons were made between littermates. Variance was reported as SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Aß40 inhibits amyloid deposition in bitransgenic BRI-Aß40/Tg2576 mice
We crossed hemizygous BRI-Aß40 mice that produce only Aß40 with hemizygous APPswe (Tg2576) mice overexpressing a normal profile of human Aß peptides (Hsiao et al., 1996), generating offspring with nontransgenic (non-Tg), BRI-Aß40, Tg2576, and BRI-Aß40/Tg2576 genotypes. The extent of parenchymal and vascular Aß deposition in an aging series of littermates (8–20 months of age) was analyzed by biochemical and immunohistochemical methods. The forebrain and hindbrain were analyzed independently because of regional differences in Aß production between BRI-Aß mice and Tg2576 mice (McGowan et al., 2005). As noted previously, BRI-Aß40 mice did not develop amyloid pathology at any age (Fig. 1C,D) (McGowan et al., 2005). Surprisingly, BRI-Aß40/Tg2576 mice had dramatic (60–90%) reductions in both immunohistochemical Aß loads and ThioS-positive plaques compared with age-matched Tg2576 littermates (Fig. 1). Likewise, biochemical analyses of Aß levels showed 60–80% reductions in RIPA-insoluble, formic acid (FA)-extractable Aß40 and Aß42 levels in the forebrain and hindbrain of the bitransgenic mice (Fig. 2), although steady-state soluble Aß (Aß40 plus Aß42) levels were increased by approximately twofold to fourfold in bitransgenic mice compared with Tg2576 littermates (see Fig. 5A,B). Leptomeningeal CAA, with a typical concentric Aß immunostaining pattern, was also reduced in BRI-Aß40/Tg2576 mice compared with Tg2576 littermates (Fig. 3). The CAA severity score and the number of CAA-affected leptomeningeal vessels per section were decreased by 60 and 30% at 15 and 20 months of age, respectively (Fig. 3C,D).

View larger version (57K): [in this window] [in a new window]   Figure 1. Decreased amyloid deposition in BRI-Aß40/Tg2576 mice. A, B, Representative entorhinal/piriform cortex sections from Tg2576 and BRI-Aß40/Tg2576 mice at 15 (A) and 20 (B) months of age were immunostained with 33.1.1 (anti-Aß1-16; top panels) or stained with ThioS (bottom panels). Scale bars: (in A) top panels, 200 µm; bottom panels, 50 µm. C, D, The amyloid plaque burden and number of ThioS-positive cored plaques in the entorhinal/piriform cortex (Ctx) and hippocampus (Hip) at 15 (C) and 20 (D) months were quantified. There was a significant decrease in both the Aß plaque burden and number of ThioS-positive plaques in BRI-Aß40/Tg2576 mice compared with Tg2576 littermates (p < 0.05). For statistical analysis, see Materials and Methods.

 

View larger version (8K): [in this window] [in a new window]   Figure 2. Decreased Aß accumulation in BRI-Aß40/Tg2576 mice. A, RIPA-insoluble, FA-extractable Aß40 and Aß42 levels in the forebrain of BRI-Aß40/Tg2576 were significantly reduced compared with Tg2576 littermates at all ages (Aß40 levels at p = 0.01, p < 0.001, and p < 0.001 at 11, 15, and 20 months, respectively, and Aß42 levels at p < 0.001 at all age). There was no evidence for accumulation of insoluble Aß in the BRI-Aß40 mice at any age. B, Because Tg2576 have only minimal accumulation of FA-extractable Aß up to 15 months of age, comparisons of FA–Aß levels in BRI-Aß40 x Tg2576 progeny were determined at 20 months of age. There was an 80% reduction in FA–Aß42 in the hindbrain of bitransgenic BRI-Aß40/Tg2576 mice compared with single transgenic Tg2576 littermates (p = 0.022 by rank sum test). Decreased FA–Aß40 levels were also detected but did not reach statistical significance (p = 0.101 by rank sum test). M, Months.

 

View larger version (43K): [in this window] [in a new window]   Figure 3. Increased Aß40 levels reduce congophilic amyloid angiopathy. A, B, CAA in cortical leptomeningeal vessels immunostained with a biotinylated-Ab9 antibody was shown in Tg2576 (A) and BRI-Aß40/Tg2576 (B) mice at 15 months of age. C, D, At both 15 (C) and 20 (D) months of age, there was a decrease in both the CAA severity score and number of CAA-affected vessels in BRI-Aß40/Tg2576 mice compared with Tg2576 age-matched littermates (p < 0.05; t test).

 
Aß40 inhibits amyloid deposition in bitransgenic BRI-Aß40/BRI-Aß42A mice
Next we examined Aß deposition in bitransgenic BRI-Aß40/BRI-Aß42A mice produced by crossing hemizygous BRI-Aß40 mice with hemizygous BRI-Aß42A mice. BRI-Aß42A mice initially develop amyloid deposition in the cerebellum at 3 months of age, whereas forebrain pathology was consistently observed only after 12 months of age (McGowan et al., 2005). Extensive premature death observed in BRI-Aß40/BRI-Aß42A mice limited rigorous pathological analyses to an 8 month time point (see Fig. 7A). At this age, Aß plaque burden, the number of ThioS-positive cored plaques in the cerebellum, and FA-fraction Aß42 levels were significantly decreased by 75% in BRI-Aß40/BRI-Aß42A mice (Fig. 4), although steady-state soluble brain Aß (Aß40 plus Aß42) levels were increased by 10-fold in bitransgenic mice compared with BRI-Aß42A littermates (Fig. 5D). BRI-Aß40/BRI-Aß42A mice also had markedly less CAA than BRI-Aß42A littermates (Fig. 4D). The results from BRI-Aß40/BRI-Aß42A mice confirmed our findings from BRI-Aß40/Tg2576 mice, indicating Aß40 inhibited amyloid deposition in vivo.

View larger version (36K): [in this window] [in a new window]   Figure 4. Decreased amyloid deposition in BRI-Aß40/BRI-Aß42A mice. A, Serial cerebellar sections from 8-month-old mice were immunostained with 33.1.1 (anti-Aß1-16; top panels) and stained with ThioS (bottom panels). Scale bar: top panels, 200 µm; bottom panels, 50 µm. B, Both the Aß plaque burden (p = 0.007; t test) and the number of ThioS-positive plaques (p < 0.001; t test) were significantly reduced in BRI-Aß40/BRI-Aß42A mice compared with age-matched BRI-Aß42A littermates. C, Similarly, RIPA-insoluble, FA-extractable Aß42 levels in the cerebellum of BRI-Aß40/BRI-Aß42A mice were markedly lower compared with BRI-Aß42A littermates (p = 0.01; t test). D, Both the severity of CAA and the number of CAA-affected vessels in cerebellar leptomeninges were reduced in BRI-Aß40/BRI-Aß42A mice compared with BRI-Aß42A mice (p < 0.001; rank sum test). There was no amyloid pathology, CAA, or accumulation of RIPA-insoluble FA–Aß in BRI-Aß40 mice.

 

View larger version (11K): [in this window] [in a new window]   Figure 5. Steady-state RIPA-soluble brain Aß levels and plasma Aß levels in BRI-Aß40/Tg2576 and BRI-Aß40/BRI-Aß42A mice before amyloid deposition. To ensure that there was no change in transgene expression levels or alteration in production of Aß levels in any of the bigenic mice, RIPA-soluble Aß levels in forebrain, hindbrain, and plasma were analyzed by Aß sandwich ELISAs. A–C, The levels of RIPA-soluble Aß40 and Aß42 in forebrain (A), hindbrain (B), and plasma (C) of BRI-Aß40/Tg2576 mice were consistent with an additive sum of Aß levels from their single transgenic littermates at 8 months of age (p > 0.1). D, E, Bitransgenic BRI-Aß40/BRI-Aß42A mice had comparable Aß40 and Aß42 levels in hindbrain (D) and plasma (E) compared with BRI-Aß40 and BRI-Aß42A single transgenic littermates at 2.5 months of age, respectively (p > 0.1). For statistical analysis, see Materials and Methods.

 
No alteration in steady-state soluble Aß levels before amyloid deposition
Because reduced Aß deposition in BRI-Aß40/Tg2576 and BRI-Aß40/BRI-Aß42A mice might be attributable to effects of transgene expression and/or Aß production and because these would be reflected by changes in steady-state Aß levels, we measured RIPA-soluble brain Aß levels and plasma Aß levels before the significant accumulation of Aß in the brain. RIPA-soluble Aß levels in the brain and plasma of BRI-Aß40/Tg2576 mice were consistent with an additive sum of soluble Aß levels of their single transgenic littermates at 8 months of age (Fig. 5A–C). Indeed, these data demonstrate that the large decrease in amyloid deposition is attributable to a doubling of Aß40 levels in BRI-Aß40/Tg2576 mice (Fig. 5A). Similarly, BRI-Aß40/BRI-Aß42A mice had no significant differences in soluble Aß40 and Aß42 levels compared with BRI-Aß40 and BRI-Aß42A littermates, respectively (Fig. 5D,E). These results confirm that the reduced amyloid deposition observed in BRI-Aß40/Tg2576 mice and BRI-Aß40/BRI-Aß42A mice was not caused by decreased steady-state soluble Aß levels.

No alteration in APP processing in bitransgenic BRI-Aß40/Tg2576 mice
Recently, an interaction between BRI and wild-type APP (APP_wt) was reported in vitro. The binding of BRI to APP_wt resulted in decreased Aß and increased C99 levels, with inconclusive effects of BRI on full-length APP and total secreted APP levels (Fotinopoulou et al., 2005; Matsuda et al., 2005). As noted above, we saw no evidence for altered production of Aß in the crossed mice. To further investigate the possible inhibition of APP processing by BRI-Aß40 protein in the transgenic mice, we examined C99 and APP protein levels by Western blot analysis (Fig. 6A,B). Steady-state C99 and APP protein levels were unchanged between the Tg2576 mice and BRI-Aß40/Tg2576 mice (Fig. 6C,D). These results together with the data from the bitransgenic BRI-Aß40/BRIAß42 mice indicate that the reduced amyloid burden and decreased accumulation of FA-fraction Aß are not attributable to interference in APP processing by the BRI transgene.

View larger version (14K): [in this window] [in a new window]   Figure 6. No alteration in amyloidogenic APP processing in BRI-Aß40/Tg2576 mice. RIPA-soluble forebrain extracts from 8-month-old BRI-Aß40/Tg2576, Tg2576, BRI-Aß40, and non-Tg littermates were analyzed by Western blotting. A, B, Blots were probed with 82E1 (anti-Aß1-16) (A) or CT20 (anti-APP C-terminal 20 amino acids) (B), stripped, and reprobed with anti-ß actin to assess loading. C, D, The relative levels of C99 (C) and APP (D) after normalization to ß-actin were equivalent between Tg2576 mice and BRI-Aß40/Tg2576 mice, indicating that there was no alteration in the processing of APP in the bitransgenic mice (p > 0.1). C99 and APP levels were equivalent between non-Tg and BRI-Aß40 mice that express only endogenous mouse APP (p > 0.1). n = 5 mice per genotype. For statistical analysis, see Materials and Methods.

 
Aß modulates premature-death phenotype
In contrast to many lines of mutant APP mice that exhibit a premature-death phenotype (Moechars et al., 1999b; Leissring et al., 2003), BRI-Aß40 or BRI-Aß42 mice did not have accelerated mortality (Fig. 7A). Surprisingly, BRI-Aß40/BRI-Aß42A mice had a progressive premature-death phenotype that approached 100% death by 16 months of age (p < 0.001; compared with singly transgenic and non-Tg littermates) (Fig. 7A). When BRI-Aß40 mice were crossed with a second line of BRI-Aß42B mice that express lower levels of Aß42 (50% less than BRI-Aß42A line), bitransgenic BRI-Aß40/BRI-Aß42B mice still died prematurely, although at a slower rate than BRI-Aß40/BRI-Aß42A mice. BRI-Aß42A/Tg2576 mice exhibited an enhanced premature-death phenotype (50%) relative to Tg2576 littermates (30%), whereas BRI-Aß40/Tg2576 mice had a significantly reduced death rate (10%) compared with their Tg2576 littermates at 7 months of age (Fig. 7B).

View larger version (11K): [in this window] [in a new window]   Figure 7. Aß modulates premature death. A, Survival rates for progeny of BRI-Aß40 mice bred with BRI-Aß42A mice (highest-expressing Aß42 line) and BRI-Aß42B mice (lower-expressing Aß42 line) were calculated using Kaplan–Meier methods. Bitransgenic BRI-Aß40/BRI-Aß42A had an accelerated mortality, whereas non-Tg, single transgenic BRI-Aß40, and BRI-Aß42A mice did not show any premature death (p < 0.01). BRI-Aß40/BRI-Aß42B mice had a decreased premature death rate compared with BRI-Aß40/BRI-Aß42A mice (p < 0.01). B, Kaplan–Meier survival curves for progeny of BRI-Aß40 and BRI-Aß42A crossed with Tg2576 mice. BRI-Aß42A/Tg2576 mice had a significantly increased premature death rate compared with Tg2576 littermates (p < 0.05). Although BRI-Aß40/Tg2576 only had 10% premature death at 7 months of age, Tg2576 mice had 30% early death (p < 0.01). There was no difference in survival rates for Tg2576 progeny from breeding with either BRI-Aß40 mice or BRI-Aß42A mice (p = 0.884), thus Tg2576 data were pooled. However, only littermates from breeding experiments were used for multiple comparison tests. For statistical analysis, see Materials and Methods.

 
Aß40 inhibits Aß42 aggregation in vitro
To understand the underlying mechanism by which Aß40 reduced amyloid deposition in BRI-Aß40/Tg2576 and BRI-Aß40/BRI-Aß42A mice, we determined whether Aß40 could directly inhibit Aß42 fibrillogenesis in vitro using an Aß aggregation assay. When freshly prepared Aß42 mixtures were incubated for 2 h, all Aß42 aggregated as high molecular complexes (Fig. 8). However, addition of Aß40 to Aß42 preparation led to a reduction in high molecular complex formation, and most Aß42 still remained as low molecular weight species (Fig. 8). These nondenaturing electrophoresis results indicate that Aß40 directly inhibits the Aß42 aggregation process in vitro.

View larger version (11K): [in this window] [in a new window]   Figure 8. Aß40 inhibits Aß42 aggregation in vitro. Synthetic Aß40 or Aß42 peptides were mixed at various molar ratios (1:1.5 and 0.5:0.75 µM). Aß mixtures were either directly used for analysis (indicated by "–") or incubated for 2 h at 37°C (indicated by "=") and electrophoresed on 4–20% Tris-HCl gels under nondenaturing conditions. The blot was probed with Ab9. After a 2 h incubation, the Aß42-only preparation (Aß40:Aß42 ratio; 0:1) formed very high molecular weight (HMW) aggregates (indicated by "" at the top of blot) without any low molecular weight LMW species, whereas the Aß40 only preparation (Aß40:Aß42 ratio; 1:0) remained as a LMW species (indicated by at the bottom of blot). When Aß40 was mixed with Aß42 (Aß40:Aß42 ratio; 0.5:1 and 1:1), Aß40 inhibited the formation of HMW Aß42 aggregates, and the vast majority of Aß42 remained as LMW species (indicated by at the bottom of the blot).

 

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our surprising results unequivocally demonstrate that Aß40 has a strong anti-amyloidogenic effect in vivo; increasing Aß40 levels in the brain of Tg2576 or BRI-Aß42A mice protected against amyloid pathology. Moreover, the magnitude of this effect is quite unexpected: approximately twofold increases of Aß40 levels in the forebrain of the BRI-Aß40/Tg2576 mice had a lifelong inhibitory effect on Aß deposition ranging from 80% reduction at 11 months to 50% at 20 months, compared with Aß deposition in Tg2576 littermates. By inference, decreasing Aß40 levels should increase amyloid pathology. Several studies do support the notion that decreasing Aß40 levels exacerbates the AD phenotype. Decreases in Aß40, without an increase in Aß42, have been associated with a subset of AD causing PSEN mutations and the APPV715M mutation (Ancolio et al., 1999; Bentahir et al., 2006; Kumar-Singh et al., 2006). In addition, several transgenic modeling studies support the notion that Aß40 may be protective. Small reductions in brain Aß40 levels, with no change in Aß42 levels, resulting from expression of an artificial exon 10 deletion mutant PSEN1 transgene were also associated with exacerbated amyloid plaque pathology in Tg2576 mice (Deng et al., 2006). Finally, results from transgenic mice expressing wild-type and various mutant forms of APP also suggest that increased Aß40 levels might reduce amyloid deposition (Mucke et al., 2000).

Because these studies rely on comparisons of Aß deposition between wild-type and mutant APP transgenic mice or APP mice crossed with wild-type and artificial mutant PSEN transgenic mice, there are numerous confounds that prevent definitive assertions regarding the role of Aß40. Mutations in APP and PSEN can alter production of not only Aß42 but also other Aß peptides (e.g., Aß1-38) and both the levels of APP processing derivatives and the subcellular localization of processing. In addition, mutations in PSEN can have a variety of effects on protein trafficking, clearance, and intracellular signaling (Koo and Kopan, 2004; Zhang et al., 2006). These complicating factors are mostly avoided in our study by using the BRI-Aß fusion system. Selective expression of Aß1-40 decreased Aß deposition in Tg2576 mice, whereas selective expression of Aß1-42 in Tg2576 mice had the exact opposite effect on the pathology of plaques (McGowan et al., 2005).

Although there is much debate regarding the role of amyloid in AD pathogenesis (Le et al., 2001; Caughey and Lansbury, 2003; D'Amore et al., 2003; Lombardo et al., 2003; Tsai et al., 2004), an increasing body of evidence suggests that soluble oligomeric and protofibrillar Aß species, such as Aß*56, may cause the synaptic dysfunction and memory deficits in mice (Klein et al., 2004; Cleary et al., 2005; Glabe, 2006; Lesne et al., 2006). Therefore, the effect of increasing Aß40 levels on oligomer formation and behavioral abnormalities in Tg2576 mice requires additional investigation.

There are several in vitro studies demonstrating that Aß40 directly interferes with Aß42 aggregation by delaying the Aß42-mediated nucleation step at an early stage in the fibrillogenesis process (Snyder et al., 1994; Hasegawa et al., 1999; Zou et al., 2003). Additional studies have shown that wild-type Aß40 can stabilize aggregation of Arctic mutant Aß40 (E22G) (Lashuel et al., 2003). Our results from an in vitro Aß aggregation assay confirm these previous studies, indicating that a direct inhibitory effect of Aß40 on Aß42 aggregation into amyloid is the most likely mechanism that accounts for our in vivo findings.

Increased Aß40 levels in BRI-Aß40/Tg2576 and BRI-Aß40/BRI-Aß42A mice led to a reduction in CAA, demonstrating that Aß40 has anti-amyloidogenic effects on not only parenchymal but also vascular amyloid deposition. This is a surprising result given that Aß40 is reported to be the predominant Aß species deposited in vessels (Gravina et al., 1995). Although previous studies suggested that a higher ratio of Aß40 to Aß42 might promote the formation of CAA over parenchymal deposition (Herzig et al., 2004; Fryer and Holtzman, 2005; Fryer et al., 2005), in other studies, selective increases in Aß42 were associated with more CAA (Van Dorpe et al., 2000; Samura et al., 2006; Van Dooren et al., 2006). Our previous studies showed that high-level production of wild-type Aß40 by itself is not sufficient to cause CAA (McGowan et al., 2005). In any case, additional studies will be needed to understand the factors that promote Aß40 accumulation within vessels in AD.

Premature death has been observed in many mutant APP transgenic mice on multiple background strains, although no one has been able to determine the cause of death (Hsiao et al., 1995; Moechars et al., 1999b; Leissring et al., 2003). BRI-Aß40/BRI-Aß42A mice had a progressive and ongoing death rate, whereas mortality in BRI-Aß40/Tg2576 and BRI-Aß42A/Tg2576 mice stabilized after 6 months of age. Premature death occurs well before plaque deposition in BRI-Aß40/BRI-Aß42A mice, implying that early high mortality is not directly associated with plaque formation as reported previously (Moechars et al., 1999a,b; Leissring et al., 2003).

Bitransgenic BRI-Aß40/Tg2576 mice had a significantly reduced premature death rate with a concomitant decrease in Aß deposition compared with their Tg2576 littermates. Reduction in Aß levels by increased -secretase activity or by enhanced proteolysis of Aß has been linked to prevention of high mortality in APP transgenic mice and Drosophila (Leissring et al., 2003; Etcheberrigaray et al., 2004; Finelli et al., 2004). Together, these studies provide indirect evidence that alterations in Aß levels can modulate the premature-death phenotype, although one study suggests that this is not because of Aß (Krezowski et al., 2004). Although it is difficult to completely exclude other transgene-related and genetic background effects, our data suggest that an interaction between Aß40 and Aß42 is required for the premature-death phenotype. However, the rate and extent of premature death are influenced by total levels of Aß, the ratio of Aß40 to Aß42, and the genetic background.

The inhibition of amyloid deposition by Aß40 may have critical implications for AD therapy. Our data support the strategy that selectively targeting Aß42 by allosterically modulating -secretase may be preferential to nonselective inhibition of -secretase activity (Weggen et al., 2001; Eriksen et al., 2003; Lleo et al., 2004). Indeed, strategies that preferentially target Aß40 production, as some -secretase inhibitors do, could exacerbate amyloid deposition. Notably, there are several other examples of anti-aggregation effects of homologous proteins. ß-Synuclein inhibits -synuclein aggregation in mice, and mouse tau may retard human tau aggregation (Rochet et al., 2000; Hashimoto et al., 2001; Andorfer et al., 2003). Thus, a common mechanism underlying many neurodegenerative diseases characterized by accumulation of misfolded proteins may be an imbalance between pro-amyloidogenic (i.e., Aß42 and -synuclein) and anti-amyloidogenic (i.e., Aß40 and ß-synuclein) proteins.

	Footnotes

 
Received Nov. 7, 2006; accepted Dec. 10, 2006.

This work was supported by National Institute on Aging Grant RO1 AG022595 to E.M. Additional resources from the Mayo Foundation, provided by a gift from Robert and Clarice Smith, were used to support the Tg2576 mouse colony. We thank Dr. J. Crook and M. Heckman for advice on statistical analysis; Dr. T. Rosenberry for valuable discussion; F. Conkle and the Veterinary Medicine staff for animal maintenance; L. Rousseau, V. Phillips, and M. Castanedes-Casey for expert histology; and Drs. J. Eriksen and C. Zehr for Metamorph programming.

Correspondence should be addressed to Dr. Eileen McGowan, Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San Pablo Road, Jacksonville, FL 32224. Email: mcgowan.eileen{at}mayo.edu

Copyright © 2007 Society for Neuroscience 0270-6474/07/270627-07$15.00/0

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