Abstract
A cardinal pathological lesion of Alzheimer's disease (AD) is the deposition of amyloid β (Aβ) in the brain. We previously reported that exposing transgenic mice harboring APPswe/PS1ΔE9 transgenes to an enriched environment resulted in reduced levels of Aβ peptides and deposition, findings that were correlated with an increase in the expression of TTR, encoding transthyretin (TTR). TTR is expressed at high levels in the choroid plexus and known to bind Aβ peptides and modulate their aggregation in vitro and in vivo. To explore the impact of TTR expression on Aβ levels and deposition in vivo, we crossed ceAPPswe/PS1ΔE9 transgenic mice to mice with genetic ablations of TTR. We now report that the levels of detergent-soluble and formic acid-soluble levels of Aβ and deposition are elevated in the brains of ceAPPswe/PS1ΔE9/TTR+/− mice compared with age-matched ceAPPswe/PS1ΔE9/TTR+/+ mice. Moreover, Aβ deposition is significantly accelerated in the hippocampus and cortex of ceAPPswe/PS1ΔE9/TTR+/− mice. Our results strongly suggest that TTR plays a critical role in modulating Aβ deposition in vivo.
Introduction
Alzheimer's disease (AD) is associated with progressive memory loss and severe cognitive decline. These clinical features are associated with deposition of 40–42 amino acid β-amyloid (Aβ) peptides in the cerebral cortex and hippocampal formation. Aβ peptides are liberated from amyloid precursor proteins (APP), by the concerted action of BACE 1 (Vassar et al., 1999; Yan et al., 1999) and “γ”-secretase (Sisodia and St George-Hyslop, 2002; De Strooper, 2003). Early onset, familial forms of the disease (FAD) are caused by expression of mutant variants of APP, presenilin 1 (PS1), or presenilin 2 (PS2) (Price and Sisodia, 1998).
We previously reported that APPswe/PS1ΔE9 transgenic mice exposed to an “enriched” environment exhibited reduced Aβ in the cortex and hippocampus compared with APPswe/PS1ΔE9 mice maintained in standard housing conditions (Lazarov et al., 2005). Extending these findings, our high density DNA microarray profiling studies revealed that expression of TTR, a gene encoding transthyretin (TTR), was significantly upregulated in the brains of the enriched APPswe/PS1ΔE9 transgenic mice. TTR, a homoterameric protein of 127 amino acid subunits, is synthesized in the liver and by epithelial cells of the choroid plexus (CSF). Serum TTR is involved in the transport of thyroxine (Schreiber et al., 1990; Chanoine et al., 1992) and plasma retinol-binding protein complexed to vitamin A (Monaco, 2000). A series of biochemical and in vivo studies have revealed that TTR may also play a role in modulating Aβ aggregation both in vitro and in vivo. For example, Aβ forms stable complexes with TTR in vitro and prevents aggregation/amyloid formation (Schwarzman et al., 1994), whereas expression of human TTR in Caenorhabditis elegans rescues the morphological and behavioral alterations in worms expressing human Aβ peptides in the muscle (Link, 1995). Finally, microarray studies of hippocampi from 6-month-old Tg2576 transgenic mice (Stein and Johnson, 2002), or cortical tissue from Tg2576/PS1P264L/P264L mice analyzed well before the onset of Aβ deposition (Wu et al., 2006), have revealed markedly elevated levels of TTR transcripts. These studies suggested that TTR gene expression was induced in response to overproduction of Aβ peptides (Stein and Johnson, 2002) and that overexpressed TTR would sequester Aβ species and thus preclude their subsequent aggregation and deposition.
To explore the impact of TTR expression on Aβ levels and deposition in vivo, we crossed mice that harbor FAD-linked APPswe and PS1ΔE9 transgenes (Jankowsky et al., 2001) to mice with homozygous deletions of TTR. Brain Aβ levels and amyloid deposition in ceAPPswe/PS1ΔE9/TTR+/+ or ceAPPswe/PS1ΔE9/TTR+/− mice were examined as a function of age. We now report that amyloid deposition is accelerated and Aβ levels are significantly elevated in the brains of ceAPPswe/PS1ΔE9/TTR+/− compared with ceAPPswe/PS1ΔE9/TTR+/+ mice at all ages examined. Our results strongly suggest that TTR plays a critical role in modulating Aβ deposition in vivo.
Materials and Methods
Transgenic mice.
The ceAPPswe/PS1ΔE9 transgenic mouse line #57 (Jankowsky et al., 2001) was obtained from The Jackson Laboratory (Bar Harbor, ME). Mice with a targeted insertion into exon 2 of the TTR gene (Episkopou et al., 1993) were obtained from Dr. William Blaner (Columbia University, New York, NY).
Tissue processing.
The mice were deeply anesthetized with a mixture of ketamine and xylazine and then decapitated. Isolated brains were bisected longitudinally, and hemispheres were separated and frozen on dry ice. The left hemisphere was used for examination of cerebral Aβ quantification by ELISA or protein expression by Western blot analysis. The right hemisphere was kept intact for immunohistochemistry (see below).
Sandwich ELISA analysis, immunocytochemistry, and Western blot analysis.
The levels of cerebral Aβ were detected using ELISA protocols described previously (Suzuki et al., 1994; Turner et al., 1996). Immunostaining of sections, confocal imaging, and quantification of amyloid deposition were performed as described by Lazarov and et al. (2005). For Western blot analysis, brains were homogenized in detergent-containing buffer and subject to Western blot analysis as described previously (Lazarov et al., 2005). APP and APP-C-terminal fragments (CTFs) were detected using Ab369, raised against the APP C terminus (Xu et al., 1997). Full-length and soluble APP derivatives were detected with Ab22C11, a monoclonal antibody raised against the extracellular domain of APP (Hilbich et al., 1993), whereas the soluble derivative derived from APPswe was detected with Ab192swe (Haass et al., 1995). TTR was detected with rabbit anti-mouse TTR antibody (Sousa et al., 2007). Mouse anti-α-tubulin was used to normalize for protein loading.
Statistical analysis.
Data are expressed as mean values ± SEM. Student's t test and ANOVA tests were applied to study the relationship between the different variables. Values of p < 0.05 were considered to be significant.
Results
Increased levels of cerebral Aβ levels in brains of hemizygous TTR-deficient mice harboring ceAPPswe/PS1ΔE9 transgenes
The ceAPPswe/PS1ΔE9 transgenic mouse line #57 (Jankowsky et al., 2001) harbors cointegrated APPswe and PS1ΔE9 transgenes are driven by the mouse prion promoter (PrP). The APPswe transgene encodes a chimeric mouse-human APP695 harboring a human Aβ domain and mutations (K595N, M596L) linked to Swedish FAD pedigrees (APPswe), and the human PS1ΔE9 transgene is linked to familial AD (Borchelt et al., 1996, 1997; Lee et al., 1997). The bigenic ceAPPswe/PS1ΔE9 were crossed with TTR-deficient mice (Episkopou et al., 1993) to generate ceAPPswe/PS1ΔE9/TTR+/− or ceAPPswe/PS1ΔE9/TTR+/+ mice. Although we obtained a number of ceAPPswe/PS1ΔE9/TTR−/− in the course of breeding, we chose not to evaluate Aβ levels and deposition in these animals to avoid indirect effects of TTR-deficiency that could confound our interpretations. For example, TTR-deficient mice exhibit significantly elevated expression of mRNA encoding peptidylglycine α-amidating monooxygenase, the rate-limiting enzyme in neuropeptide maturation, and hence, leads to elevated levels of neuropeptide Y (NPY) (Nunes et al., 2006). NPY acts on energy homeostasis by increasing white adipose tissue lipoprotein lipase and decreasing thermogenesis. Indeed, TTR-deficient mice exhibit decreased body temperature, increased carbohydrate consumption, and preference (Nunes et al., 2006), and we observed that both TTR−/− and ceAPPswe/PS1ΔE9/TTR−/− mice are lethargic and exhibit increased body weight relative to their hemizygous TTR (or ceAPPswe/PS1ΔE9/TTR+/−) or TTR+/+ (or ceAPPswe/PS1ΔE9/TTR+/+) littermates (S.H.C. and S.S.S., personal observations). In addition, the limbic forebrain of TTR-deficient mice exhibits significantly elevated levels of noradrenaline (Sousa et al., 2004), a catecholamine neurotransmitter that has been shown to modulate Aβ burden in a transgenic mouse model of AD (Kalinin et al., 2007). Finally, because retinol and thyroid hormones are essential for normal mammalian brain physiology and are particularly critical during development (Porterfield and Hendrich, 1993) and because TTR is the only thyroid hormone-binding protein found at a substantial level in the CSF (Herbert et al., 1986), it is possible that TTR reduction could cause developmental abnormalities.
To examine the influence of TTR expression on cerebral steady-state levels of Aβ, we generated mice that harbored ceAPPswe/PS1ΔE9 transgenes on a TTR+/− background. Cohorts of these animals were aged for either 3, 4, 5, or 7 months. Aβ levels in detergent and formic acid extracts of hemibrains of either ceAPPswe/PS1ΔE9/TTR+/+ or ceAPPswe/PS1ΔE9/TTR+/− mice were quantified using sandwich ELISA analysis. We show that 5-month-old ceAPPswe/PS1ΔE9/TTR+/− mice exhibit elevated steady-state levels of detergent soluble AβX-40 and AβX-42 compared with 5-month-old ceAPPswe/PS1ΔE9/TTR+/+ mice (Fig. 1A and B, respectively), but the differences failed to reach significance. In contrast, the levels of detergent soluble AβX-40 and AβX-42 peptides were significantly elevated in the brains of 7-month-old ceAPPswe/PS1ΔE9/TTR+/− compared with ceAPPswe/PS1ΔE9/TTR+/+ mice (Fig. 1A,B). These studies suggested that a fraction of Aβ peptides that would otherwise aggregate and deposit remain in a detergent extractable state when TTR gene dosage is reduced. Most notably, the levels of formic acid-soluble AβX-40 (Fig. 1C) and AβX-42 (Fig. 1D) peptides were significantly elevated in the brains of the ceAPPswe/PS1ΔE9/TTR+/− compared with ceAPPswe/PS1ΔE9/TTR+/+ mice at both the 5 and 7 month time points, findings that would argue in support for a role of TTR in preventing Aβ aggregation and subsequent deposition.
Genetic reduction of TTR elevates steady-state levels of cerebral Aβ in the brains of ceAPPswe/PS1ΔE9 mice. A, B, Levels of detergent-soluble AβX-40 and AβX-42 in brain extracts of ceAPPswe/PS1ΔE9/TTR+/− and ceAPPswe/PS1ΔE9/TTR+/+ mice at 7 month time point. C, D, Levels of formic acid-soluble AβX-40 and AβX-42 in brain extracts of ceAPPswe/PS1ΔE9/TTR+/− and ceAPPswe/PS1ΔE9/TTR+/+ mice at 5 and 7 month time points. The asterisk indicates a significant difference from ceAPPswe/PS1ΔE9/TTR+/+ at p < 0.05 (3–4 animals/group). Error bars represent SE.
Genetic reduction of TTR leads to accelerated Aβ deposition in ceAPPswe/PS1ΔE9 mice
To examine the influence on reduced expression of TTR on amyloid deposition, brain sections from ceAPPswe/PS1ΔE9/TTR+/− and ceAPPswe/PS1ΔE9/TTR+/+ mice were probed with Aβ-specific 3D6 antibodies (Kim et al., 2001), and bound antibodies were detected by fluorescently labeled secondary antibodies. Confocal images of amyloid burden were quantified by morphometric methods.
Although we failed to detect any significant difference in the levels of Aβ deposition between genotypes at the 3 and 4 month time points, we observed a dramatic and significant elevation in amyloid deposition in the cortex of ceAPPswe/PS1ΔE9/TTR+/− at 5 and 7 months compared with ceAPPswe/PS1ΔE9/TTR+/+ mice (Fig. 2A, compare f and g with b and c, respectively). Similarly, amyloid deposition in the hippocampus of 7-month-old ceAPPswe/PS1ΔE9/TTR+/− was clearly elevated compared with ceAPPswe/PS1ΔE9/TTR+/+ mice (Fig. 2A, compare h and d, respectively). Morphometric analysis confirmed that ceAPPswe/PS1ΔE9/TTR+/− exhibited significantly higher amyloid burden in the cortex (Fig. 2Ba) and hippocampus (Fig. 2Bb) compared with ceAPPswe/PS1ΔE9/TTR+/+ mice at the 5- and 7-month-old time points. We then costained brain sections from 5-month-old mice with thioflavin S and 3D6 antibodies (Fig. 2C). These studies revealed that thioflavin S staining in ceAPPswe/PS1ΔE9/TTR+/− was elevated compared with the ceAPPswe/PS1ΔE9/TTR+/+ mice, and that this staining was at the core of the amyloid deposits in both cases.
Increased amyloid deposition in the cortex and hippocampus of ceAPPswe/PS1ΔE9/TTR+/−. A, Immunohistochemical analysis of brain sections of APPswe/PS1ΔE9/TTR+/+ (a–c, cortex, 4, 5, and 7 month; d, hippocampus, 7 month) and ceAPPswe/PS1ΔE9/TTR+/− (e, f, cortex, 4, 5, and 7 month; h, hippocampus, 7 month) mice immunolabeled with anti-Aβ 3D6 antibodies. Scale bar, 200 μm. B, Quantitative analysis of volume of amyloid burden in the cortex (a) and the hippocampus (b) of APPswe/PS1ΔE9/TTR+/− versus APPswe/PS1ΔE9/TTR+/+ mice. Volume is in arbitrary units (mean voxel count ± SE). The asterisk indicates a significant difference from ceAPPswe/PS1ΔE9/TTR+/+ at *p < 0.05; **p < 0.01 (4 animals/group). Error bars represent SE. C, Thioflavine S-stained amyloid deposits in the cortex of 5-month-old ceAPPswe/PS1ΔE9/TTR+/− versus 5-month-old ceAPPswe/PS1ΔE9/TTR+/+ mice. Costaining of brain sections with 3D6 antibodies (a, d) and thioflavine S (b, e) and overlap (c, f). Scale bar, 50 μm.
Genetic reduction of TTR does not alter APP processing in ceAPPswePS1ΔE9 mice
To assess whether alterations in APP processing might account for the observed elevations of Aβ levels in ceAPPswe/PS1ΔE9/TTR+/−, we prepared detergent-soluble extracts from these animals and ceAPPswe/PS1ΔE9/TTR+/+ mice and subjected these preparations to Western blot analysis. To confirm the TTR genotype, extracts were probed with an anti-TTR antibody. We demonstrate that an ∼14 kDa TTR antibody-immunoreactive polypeptide is present in extracts from brains of ceAPPswe/PS1ΔE9/TTR+/−, and this species is absent in extracts from ceAPPswe/PS1ΔE9/TTR−/− mice (Fig. 3A, lanes 1 and 2, respectively). The ∼14 kDa immunoreactive species is also present in extracts from brains of ceAPPswe/PS1ΔE9/TTR+/+ mice at all ages examined (Fig. 3, lanes 3, 5, 7), and the levels are clearly lower in brains of ceAPPswe/PS1ΔE9/TTR+/− mice at all ages (Fig. 3, lanes 4, 6, 8). Analysis of Western blots using Ab369 failed to disclose any differences in the levels of full-length APP (APP-FL) or membrane-tethered APP C-terminal derivates (APP-CTFs) in extracts prepared either from ceAPPswe/PS1ΔE9/TTR+/+ (Fig. 3B, lanes 3, 5, 7) or ceAPPswe/PS1ΔE9/TTR+/− (Fig. 3B, lanes 1, 4, 6, 8) mice. Furthermore, we failed to observe any differences in total levels of full-length APP and soluble derivatives using Ab22C11, raised against the extracellular domain of APP, or soluble Swedish βAPPs detected by Ab192swe between ceAPPswe/PS1ΔE9/TTR+/+ and ceAPPswe/PS1ΔE9/TTR+/− mice at all time points. These results suggest that genetic reduction of TTR in ceAPPswe/PS1ΔE9 mice does not result in a discernable impact on APP processing at steady state.
Western blot analysis of protein extracts of brains of APPswe/PS1ΔE9/TTR+/+ (WT), APPswe/PS1ΔE9/TTR+/− (+/−), or APPswe/PS1ΔE9/TTR−/− (−/−) mice. A, Western blot analysis using anti-TTR antibody. B, APP processing. Lane 1, APP-FL; Lane 2, APP-CTFs; Lane 3, α-tubulin; Lane 4, full-length and soluble APP; Lane 5, soluble Swedish βAPPs.
Discussion
A series of preceding biochemical and transgenic mouse studies have provided compelling evidence that TTR plays a role in modulating Aβ aggregation and deposition. For example, Aβ forms stable complexes with TTR and inhibits aggregation in vitro (Schwarzman et al., 1994), and expression of human TTR rescues the morphological and behavioral alterations in C. elegans that express human Aβ (Link, 1995). Indeed, studies have revealed that the levels of brain TTR were significantly lower in human AD patients compared with age-matched controls and negatively correlated with the abundance of amyloid plaques (Serot et al., 1997; Merched et al., 1998). Supporting these lines of evidence, DNA microarray studies in transgenic mice have revealed that expression of TTR mRNA is markedly elevated well before the onset of amyloid deposition (Stein and Johnson, 2002; Costa et al., 2006), suggesting that upregulation of TTR is a physiological response to elevated Aβ levels that in turn blocks their aggregation. To these studies, we demonstrated that relative to APPswe/PS1ΔE9 mice maintained in standard housing conditions, APPswe/PS1ΔE9 transgenic mice exposed to an “enriched” environment exhibited reduced Aβ deposition in the cortex and hippocampus (Lazarov et al., 2005), a setting in which steady-state levels of TTR transcripts were elevated in the brain. Collectively, these latter data provided support for the notion that TTR plays an important role in regulating Aβ deposition in vivo.
In the present study, we tested the hypothesis that lowering brain levels of TTR, a protein that can sequester Aβ peptides and prevent fibril formation, would accelerate amyloid deposition in APPswe/PS1ΔΕ9 mice, and we now offer several important insights. First, we demonstrate that the levels of detergent-soluble Aβ peptides are elevated in the brains of ceAPPswe/PS1ΔE9/TTR+/− compared with ceAPP/PS1ΔE9/TTR+/+ mice at all time points tested. Although these studies would suggest that lowering TTR levels might affect APP processing, we have not observed any alterations in APP metabolism in steady-state Western blot studies. Our interpretation of the finding of elevated soluble Aβ levels in APPswe/PS1ΔE9/TTR+/− mice is that these species are either oligomeric assemblies that are not deposited or represent the amorphous nonfibrillar assemblies that are present in the “penumbra” of the thioflavin-positive deposits. In any event, our confocal immunofluorescence and morphometric studies reveal that amyloid burden both in the cortex and hippocampus of ceAPPswe/PS1ΔE9/TTR+/− was dramatically increased compared with ceAPPswe/PS1ΔE9/+/+ mice from the 5 month time point onwards. These morphological studies were validated by sandwich ELISA analyses in which we observed that the levels of formic acid-soluble AβX-40 and AβX-42 peptides are markedly elevated in the brains of the ceAPPswe/PS1ΔE9/TTR+/− mice at all time points. Collectively, our immunohistochemical and biochemical studies convincingly demonstrate that genetic reduction of TTR elevates Aβ deposition in the brains of ceAPPswe/PS1ΔE9/TTR+/− mice.
The nature of the interaction(s) between TTR and Aβ and the mechanism(s) by which TTR alters the aggregation of Aβ in vivo are not fully understood. Liu and Murphy (2006) reported that TTR significantly decreased the rate of aggregation in a strong concentration-dependent manner. Moreover, the region near tryptophan 41 of TTR is involved in binding to Aβ aggregates by Trp fluorescence quenching experiments (Liu and Murphy, 2006), a finding consistent with studies showing that peptide fragments containing Trp41 of TTR bind to Aβ (Schwarzman et al., 2005). Although the domain(s) within Aβ that bind to TTR are not known, future efforts to obtain high-resolution information pertaining to the nature of Aβ-TTR interactions would be of considerable interest.
Finally, the sites within the brain where Aβ binds to TTR have not been fully resolved. Recent studies using laser dissection microscopy and PCR studies have clearly demonstrated that TTR transcripts are excluded from the brain parenchyma but restricted to choroid plexus (Sousa et al., 2007). These findings would argue that Aβ40/42, produced in the brain parenchyma, is subject to efflux into the CSF (Seubert et al., 1992; Shoji et al., 1992) where the peptides encounter TTR that is secreted from the choroid plexus and subsequently sequestered. Although this latter notion is attractive, it is important to note that TTR is not the only protein that binds Aβ in CSF. Indeed, evidence has accumulated that α-1-antichymotrypsin, apolipoprotein J, and apolipoprotein E, proteins present in CSF, can also bind Aβ in vitro and, in certain cases, in vivo (Abraham et al., 1988; Ghiso et al., 1993; Strittmatter et al., 1993), and the respective contributions of each of these proteins to Aβ clearance remains to be established. Notwithstanding the importance of these latter species to Aβ metabolism in vivo, our data supporting a role for TTR in modulating Aβ deposition suggest that approaches aimed at enhancing Aβ sequestration and clearance with TTR as a template would be of significant therapeutic value.
Footnotes
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This work was supported by National Institutes of Health Grants AG021494 and AG027854 (S.S.S.) and by the Edward H. Levi Fund, the Adler Foundation, and Cure Alzheimer's Fund. S.S.S. discloses that he is a paid Consultant of Neuropharma, Torrey Pines Therapeutics, and Eisai Research Labs but is not a shareholder in any company that is a maker or owner of an FDA-regulated drug or device. We thank John Robinson and Yi Huang for excellent technical assistance.
- Correspondence should be addressed to Sangram S. Sisodia, Department of Neurobiology, The University of Chicago, 947 East 58th Street, AB 308, Chicago, IL 60637. ssisodia{at}bsd.uchicago.edu
