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The Journal of Neuroscience, July 15, 2002, 22(14):5900-5909

Inflammation-Dependent Cerebral Deposition of Serum Amyloid A Protein in a Mouse Model of Amyloidosis

Jun-tao Guo1, Jin Yu1, David Grass5, Frederick C. de Beer2, and Mark S. Kindy1, 3, 4

Departments of 1 Biochemistry and 2 Internal Medicine, and 3 Stroke Program of the Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 40536, 4 Veterans Affairs Medical Center, Lexington, Kentucky 40506, and 5 Xenogen Corporation, Princeton, New Jersey 08540


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major pathological hallmark of amyloid diseases is the presence of extracellular amyloid deposits. Serum amyloid A (SAA) is an apolipoprotein primarily produced in the liver. Serum protein levels can increase one thousandfold after inflammation. SAA is the precursor to the amyloid A protein found in deposits of systemic amyloid A amyloid (AA or reactive amyloid) in both mouse and human. To study the factors necessary for cerebral amyloid formation, we have created a transgenic mouse that expresses the amyloidogenic mouse Saa1 protein in the brain. Using the synapsin promoter to drive expression of the Saa1 gene, the brains of transgenic mice expressed both RNA and protein. Under noninflammatory conditions, transgenic mice do not develop AA amyloid deposits in the brain; however, induction of a systemic acute-phase response in transgenic mice enhanced amyloid deposition. This deposition was preceded by an increase in cytokine levels in the brain, suggesting that systemic inflammation may be a contributing factor to the development of cerebral amyloid. The nonsteroidal anti-inflammatory agent indomethacin reduced inflammation and protected against the deposition of AA amyloid in the brain. These studies indicate that inflammation plays an important role in the process of amyloid deposition, and inhibition of inflammatory cascades may attenuate amyloidogenic processes, such as Alzheimer's disease.

Key words: Alzheimer's disease; transgenic; inflammation; serum amyloid A; indomethacin; cytokines; microglia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Amyloidosis encompasses a diverse group of diseases and is characterized by the extracellular accumulation of fibrillar protein deposits (Sipe, 1992; Friman and Pettersson, 1996). The most studied amyloidosis is Alzheimer's disease (AD) (Selkoe, 1994). Key pathological features in the AD brain are the presence of amyloid plaques and neurofibrillary tangles accompanied by astrocytosis, microgliosis, and synapse loss (Selkoe, 1999). The plaques are composed of amyloid beta -peptides (Abeta ), 40-42 amino acid fragments derived from the beta -amyloid precursor protein (APP) (Yankner, 1996). In attempts to understand the molecular mechanisms of amyloid formation and to explain how Abeta aggregates in AD patients, investigators have developed several lines of transgenic mice that express high levels of mutant human APP (Higgins et al., 1994; Games et al., 1995; Hsiao et al., 1996; Sturchler-Pierrat et al., 1997; Hsia et al., 1999). Some of these transgenic strains develop many of the pathological hallmarks of AD, including numerous extracellular thioflavin-S-positive Abeta deposits, neuritic plaques, and microgliosis (Games et al., 1995; Hsiao et al., 1996; Sturchler-Pierrat et al., 1997). However, these transgenic models do not allow investigators to address the question of whether extracellular deposition of fibrillar Abeta in amyloid plaques is part of the biological process that cause the neuronal dysfunction and death or is merely a byproduct of this process. This question must be answered before specific therapeutic agents can be developed.

Inflammatory mechanisms may play an important role in the pathogenesis of AD (Gahtan and Overmier, 1999). Immunocytochemical analyses have found a variety of inflammatory proteins closely associated with senile plaques (Eikelenboom and Stam, 1982; Abraham et al., 1988; Rogers et al., 1992; Smith et al., 1994; Yan et al., 1996; Selkoe, 1999). Abraham et al. (1988) associated the acute phase protein alpha 1-antichymotrypsin with Abeta deposits in the brain. Based on their findings, Vandenabeele and Fiers (1991) suggested that Abeta amyloidogenesis results from an interleukin-1 (IL-1)/IL-6-mediated acute phase reaction in the brain. Recent observations have shown elevated levels in AD brains of other inflammatory protein, such as tumor necrosis factor-alpha (TNF-alpha ), macrophage-colony stimulating factor (M-CSF), heme oxygenase-1 (HO-1), IL-1, and IL-6 (Griffin et al., 1989; Bauer et al., 1991; Yan et al., 1996; Smith et al., 1998, 2000). Serum amyloid A (SAA) was found in the brains of patients with AD but not in the brains of patients with Pick's disease or Lewy body disease (Liang et al., 1997). Saa was also shown by immunocytochemical analysis to colocalize with Abeta amyloid deposits, suggesting that acute phase response may contribute to the amyloidogenic process (Kindy et al., 1999). In addition, several clinical studies have shown that the use of nonsteroidal anti-inflammatory drugs (NSAIDs) can prevent or retard the process of AD (McGeer et al., 1995; Breitner, 1996; McGeer and McGeer, 1998). Furthermore, Lim et al. (2000) showed that APP transgenic mice treated with ibuprofen suppressed plaque pathology and inflammation associated with amyloid deposits. These studies demonstrate the importance of inflammation in the development of AD.

SAA proteins, the most dramatic acute phase reactants, are associated with high-density lipoproteins. SAA proteins are normally maintained at 1-5 µg/ml in the plasma, but during an acute phase response, levels can increase to 500-1000 µg/ml (McAdam and Sipe, 1976; Hoffman et al., 1984). SAA biosynthesis takes place primarily in the liver. No SAA expression is detected in normal brain. In mice, Saa1 and Saa2 are composed of 103 residues differing by only nine substitutions (Kindy and de Beer, 1999). Chronic inflammation in the mouse induced by a modified casein solution or by amyloid enhancing factor, and silver nitrate increased Saa expression and stimulated deposition of amyloid A (AA) amyloid (Axelrad et al., 1982). Although the two Saa proteins were found circulating in nearly equal quantities, only Saa1 was selectively deposited into amyloid fibrils (Hoffman et al., 1984; Meek et al., 1986; Shiroo et al., 1987). The ability of Saa proteins to form amyloid appears to be determined by the N terminal portion of the molecule (Westermark et al., 1992). Peptides synthesized against the first 10-15 amino acids of Saa1 formed fibrils in vitro, but Saa2 peptides were incapable of fibril formation. In addition, mice injected with synthetic Saa1 peptides developed more amyloid deposits than did mice injected with Saa2 (Ganowiak et al., 1994).

To study cerebral amyloid deposition in a homologous system, we generated transgenic mice, which express the mouse Saa1 protein in the brain. Using the rat synapsin I promoter linked to the mouse genomic Saa1 gene, transgenic mice demonstrated brain expression of Saa1 RNA and protein (Howland et al., 1991). Expression of Saa1 in mouse brain alone did not result in amyloid deposition in aged mice. However, injection of mice with lipopolysaccharide and induction of a systemic acute-phase response resulted in Saa1 immunoreactivity in the cortex and hippocampus, which was positive for Congo Red and thioflavin-S staining, indicating the presence of amyloid fibrils in transgenic but not nontransgenic mice. Systemic injection of lipopolysaccharide (LPS) resulted in an increased expression of IL-1beta , IL-6, and TNF-alpha in the brain, reactive gliosis was present around the site of deposition, and apolipoprotein E and serum amyloid P component were found associated with the amyloid deposits. Treatment with the nonsteroidal anti-inflammatory agent indomethacin dramatically reduced cytokine levels as well as AA amyloid deposition in the brain. This study shows for the first time the direct involvement of inflammation in the initiation of amyloid deposition in the brain. This transgenic model provides a novel system to study the pathobiology of cerebral amyloid and to test drugs that may slow or prevent the process of amyloidogenesis. In addition, these animals can be used to help define the role of extracellular amyloidogenic proteins in neuronal and behavioral dysfunction.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression constructs and transgenic mice. The expression construct used in this study is depicted in Figure 1. The pSP72 vector (Promega, Madison, WI) was used as the background for the transgenic construct, and oligonucleotides were synthesized to generate an SfiI site that was ligated into the HpaI site of pSP72. The Simian virus40 poly(A) signal was excised from the pGH1HG vector (British Biological Laboratories, London, UK) using EcoRI and BamHI and inserted into the EcoRI-BglII site in the pSP72 vector. The BglII-XhoI fragment of the mouse Saa1 gene was removed from the genomic construct provided by Dr. Migita (Yamamoto et al., 1986), blunt-ended and cloned into the SmaI site (pSP72A1). Finally, the 4.5 kb BamHI rat synapsin I (SYNI) promoter fragment was inserted into the BamHI site of the vector pSP72A1 (Howland et al., 1991). The plasmid pSP72A2 was linearized with HindIII and SfiI, and the Saa1 containing fragment was isolated and purified using the Geneclean II Kit (Bio 101, Inc., Vista, CA). Transgenic mice were generated at Xenogen Corporation (Princeton, NJ; formerly DNX Transgenics) by injection and manipulation of mice in procedures identical to those described previously (Hogan et al., 1982). Transgenic mice were identified by PCR analysis using genomic DNA isolated from mouse tails using a QIAamp Tissue kit (Qiagen, Valencia, CA). The mouse Saa1 transgene was detected using PCR primers against the mouse Saa1 gene (5'-GAAAGCCTCCCCAATAAATG-3') and the rat synapsin I sequence (5'- TGAGAGCGCAGCTGTGCTCCT-3') resulting in a 0.6 kb fragment. The PCR reaction was performed as follows: 4 min at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 58°C and 2 min at 72°C, and 7 min at 72°C for final extension. Four, eight, and 18-month-old male and female Tg10142 Tg+ and Tg- mice were randomly selected for use in each group.



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  		Figure 1.   Saa1 transgenic construct and mice. A, Saa1 transgenic construct. A 4.5 kb BamHI fragment of the rat synapsin I promoter was linked to the 2.4 kb BglII-XhoI mouse Saa1 gene fragment that contains the entire coding region and the SV40 poly(A) consensus sequence. The short lines under the figure indicate the transgene specific primers, which span the rat synapsin I promoter and the mouse Saa1 gene. B, PCR analysis of transgenic animals. Transgene-specific primers indicated in A were used to identify the transgenic animals. The primers identified a 603 bp fragment specific to the transgenic construct. Lanes 1-8 were from weanlings from a transgenic × transgenic cross. As indicated in the figure, the transgenic animals were identified in lanes 2, 4, 6, and 8; nontransgenic animals were in lanes 1, 3, 5, and 7. One kilobase molecular weight markers are indicated (1 kb).

Northern blot analysis. Tissues from nontransgenic and transgenic mice were harvested and frozen in liquid nitrogen and stored at -80°C. RNA was isolated from tissues using TRIzol (Invitrogen, Gaithersburg, MD). Twenty micrograms of brain RNA, and in some cases heart, liver, kidney, spleen, lung, muscle, and stomach RNA were electrophoretically separated on a 1% agarose gel containing 2 M formaldehyde. After transfer to a Duralon-UV membrane (Stratagene, La Jolla, CA), the RNA was UV cross-linked to the membrane (Stratalinker; Stratagene), and the membrane was prehybridized for 2 hr in 5× Denhardt's solution, 0.1% SDS, 1 M NaCl, 0.5% deionized formamide, and salmon sperm DNA at 42°C (Kindy et al., 1987). Hybridization was performed for 18 hr at 42°C in the same solution with a random primed mouse Saa1 cDNA labeled with [32P]-dCTP (ICN Biochemicals, Costa Mesa, CA). Before autoradiography, the membrane was washed twice for 20 min at room temperature in 2× SSC and 0.1% SDS and twice for 20 min at 65°C in 1× SSC and 0.1% SDS.

Western blot analysis. Mouse brains were homogenized in TRIzol (100 mg tissue/ml TRIzol), as described by vendor and published protocols (Liang et al., 1997). The concentrations of proteins were measured using BCA reagent (Pierce, Rockford, IL). One hundred micrograms of protein were separated on reducing SDS-PAGE (5-20%). The proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) and blocked in 5% milk and 2% BSA overnight at 4°C. The membranes were first blotted with rabbit anti-mouse Saa antibodies; the secondary antibodies conjugated to horseradish peroxidase against rabbit IgG were applied (Kindy and Rader, 1998). The protein levels of Saa1 were visualized using ECL detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunohistochemistry. Mice were perfused with 4% paraformaldehyde in PBS, and the brains were fixed in 4% paraformaldehyde overnight, and transferred into 30% sucrose for 24 hr (Kindy et al., 1995). Alternatively, brains were postfixed in 10% buffered formalin and paraffin embedded. The sections from transgenic and nontransgenic mice were placed on 1% gelatin-coated slides. Sections were treated with 3% H2O2 for 30 min, and treated with normal goat serum to block nonspecific sites before overnight incubation at 4°C with the primary antibody. Peroxidase rabbit IgG kit (Vector Laboratories, Burlingame, CA) was used as recommended, with 3,3'-diaminobenzidine (DAB) as the chromagen. Primary antibodies used in this study were as follows: rabbit alpha -mouse Saa (1:1000 dilution); rabbit alpha -rat apolipoprotein E (apoE) (1:2000); rabbit alpha -mouse serum amyloid P component (1:500; Calbiochem, La Jolla, CA); rabbit alpha -mouse glial fibrillary acidic protein (GFAP; 1:1000; Sigma, St. Louis, MO), and rabbit alpha -phospho-tyrosine (1:200; PharMingen, San Diego, CA). Amyloid deposits were quantified by counting the number of Saa immunoreactive areas and image analysis using the amyloid area in each section by a computer-assisted image analysis system, consisting of a Power Macintosh computer equipped with a Quick Capture frame grabber card, Hitachi CCD camera mounted on an Olympus (Tokyo, Japan) microscope and camera stand. NIH Image Analysis Software, version 1.55 was used. The images were captured, and the total area of deposit was determined over the five sections. A single operator blinded to treatment status performed all measurements.

Congo Red staining and thioflavin-S fluorescence. To identify the amyloid deposits, mouse brain sections were deparaffinized, rehydrated, and incubated with 1% Congo Red (Sigma). After being differentiated in alkaline alcohol solution, the sections were counterstained and dehydrated. The Congo Red-positive staining was observed under polarized light. For thioflavin-S staining, sections were deparaffinized, rehydrated, and incubated in 1% thioflavin-S (Sigma) for 10 min, then decolorized in 95% ethanol and distilled water and coverslipped with Fluoromount-G (Southern Biotechnology Association, Alabaster, AL). The sections were examined with a Nikon fluorescent microscope.

Induction of acute-phase response. An acute-phase response was elicited by intraperitoneal injection of 10 µg of LPS Escherichia coli 0111:B4 (Difco Laboratories, Detroit, MI). Two groups of transgenic mice (4 and 8 months old) were injected with LPS twice a week. In each group, half of the mice were killed after 1 month injection, the other half were killed after 1 month injection plus one more month with no injection. For inhibition studies with the anti-inflammatory drug, indomethacin (30 mg/kg, Sigma) or vehicle were injected (intraperitoneally daily) for 1 or 2 months to determine the effects of NSAIDs on amyloid formation in the brain. At the end of the experiment, animals were killed, half of the brain was taken for cytokine assays described below, and the other half was drop-fixed in 4% paraformaldehyde (0.1 M PBS) and processed for immunohistochemical analysis for AA amyloid (Saa immunoreactivity and thioflavin-S reactivity).

Cytokine analysis in the brain. Sandwich ELISA kits were used to measure the levels of IL-1beta , IL-6, and TNF-alpha in the brains of nontransgenic and transgenic mice (Endogen, Woburn, MA). Polyclonal antibodies against IL-1beta , IL-6, and TNF-alpha were used to capture the cytokine at 2 µg/ml in PBS in a 96 well plate. Plates were blocked with 2% bovine serum albumin in Tris-buffered saline, pH 7.4. Samples (100 µg of total brain protein) were incubated in the 96 well plates at 4°C for 12-24 hr. Monoclonal antibodies against IL-1beta , IL-6, and TNF-alpha , respectively, were used to detect the cytokines. Development of the ELISA was performed using horseradish peroxidase-conjugated streptavidin and tetramethylbenzidine substrate (Endogen). Detection limits of the assay were <10 U/ml for IL-1beta and IL-6, and <10 pg/ml for TNF-alpha .

Analysis of cytokine mRNA RT-PCR. RNA was isolated as described above, and cDNA was synthesized from 2 µg of total RNA in a 50 µl reaction mixture containing Moloney murine leukemia virus reverse transcriptase (400 U; Invitrogen). Reactions were performed as described previously (Gatti and Bartfai, 1993; Tehranian et al., 2001). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control to ensure that equal quantities of cDNA were amplified. Reaction products were separated on 1.5% agarose gels, and band intensity was quantified by image analysis and presented as cytokine levels/GAPDH levels.

Statistical analyses. The mean and SD were determined for each set of samples (six to eight mice per group). A two-way ANOVA was performed to determine the significant differences between transgenic and nontransgenic animals (+/- LPS), comparison of IL-1beta , IL-6, and TNF-alpha in the treated and untreated groups. Post hoc comparisons were performed using Fisher's protected least significant difference. P values <0.05 were considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of SYNI-Saa1 transgenic mice

To study the mechanism of amyloidogenesis and to generate a novel brain amyloid model, the mouse Saa1 gene driven by the neuron-specific rat SYNI promoter was introduced into mice (Fig. 1A). In mice, only the Saa1 protein is selectively deposited into amyloid fibrils, and none of the Saa proteins appear to be expressed in brain (Hoffman et al., 1984; Shiroo et al., 1987). A 4.5 kb fragment of the neuron-specific promoter SYNI was used in the generation of the transgenic construct to drive mouse Saa1 gene expression in the brain. The 2.4 kb BglII to XboI fragment of the mouse Saa1 gene was excised from a Balb/c genomic clone and was inserted into the vector between the SYNI promoter and the SV40 polyadenylation site. The construct was linearized with HindIII and SfiI, and injection was done as described previously (Hogan et al., 1982). All the founder mice appeared to develop normally, and the two lines we chose for further studies bred successfully. Transgenic offspring were screened by PCR, using primers specific for the transgene spanning the synapsin promoter and Saa1 gene (Fig. 1A). A band of 603 bp was seen in transgenic mice (Fig. 1B).

Saa1 expression in brain tissue of SYNI-Saa1 transgenic mice

Northern blot analysis showed high-level expression of the Saa1 gene in the brains in all transgenic lines (Fig. 2A), however, no Saa1 expression was seen in control littermates as reported in previous studies (Meek and Benditt, 1986). Saa1 expression was only detected in the brain of transgenic mice, not in heart, lung, kidney, spleen, muscle, stomach, and other tissues (Fig. 2B). Because liver is the major site of SAA synthesis, the constitutively expressed Saa4 and low levels of acute-phase Saa (Saa1 and Saa2) were detected in both transgenic and nontransgenic mice, and transgenic and nontransgenic mice showed the same level expression of Saa mRNA in liver. These results suggest that Saa1 was expressed only in transgenic mouse brain and not in other tissues. Immunoblot analysis of brain homogenates using anti-mouse Saa polyclonal antibodies revealed that Saa1 was expressed in several transgenic lines, but nontransgenic mice did not show any Saa protein (Fig. 2C). Unfortunately, the high-expressing lines could not breed and ultimately were lost.



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  		Figure 2.   Saa transgene expression, tissue distribution, and protein levels. A, Northern blot analysis of brain RNA from transgenic and nontransgenic animals. The expression of Saa1 mRNA was detected in the brain of transgenic animals (+) and not in the brains of nontransgenic animals (-). Expression levels varied depending on the transgenic line. Twenty micrograms of total RNA were electrophoresed onto agarose gels. Line 10142 (underlined) was selected for further analysis. B, Tissue-specific expression of the transgenic Saa1 transcript. Northern blot analysis of the Saa1 gene was performed on 20 µg of RNA from the tissues indicated. Transgenic animals had high levels of Saa1 mRNA in the brain compared with nontransgenic littermates. The low level expression of endogenous Saa1 (possibly Saa4) was detected in the liver of both transgenic and nontransgenic animals with very little, if any, detected in the other organs. C, Western blot analysis of Saa1 protein in the brains of transgenic and nontransgenic animals. Lanes 1-3, Extracts from wild-type animals. Lanes 4-6, Different transgenic lines that express Saa1. Bands were visualized with a rabbit anti-mouse Saa antibody directed against the acute-phase Saa proteins.

AA amyloid deposition in SYNI-Saa1 transgenic brain

Transgenic line 10142 was used for the following experiments (Fig. 2A,C, lane 6). Animals were killed at 4, 8, and 18 months of age to determine the amount of amyloid deposited in the brain. No amyloid deposition was detected in transgenic mice up to ~18 months of age (data not shown), although Saa1 protein was expressed in transgenic brains. These results suggested that Saa1 protein expression alone is not sufficient for amyloid formation in transgenic mice.

To assess whether a systemic acute-phase response contributes to the amyloid deposition process in the brain, both transgenic and nontransgenic mice were subject to an acute phase response by intraperitoneal LPS injections, as described in Materials and Methods. Transgenic and nontransgenic mice (8 months old) were divided into two groups. In the first group of animals, mice were injected with LPS (10 µg/mouse) twice a week for 1 month and killed after the last week. In second group, mice were injected with LPS as described for the first group, except that the mice were killed 1 month after the last set of LPS injections. Immunohistochemical analysis with anti-Saa antibodies in brains from nontransgenic mice showed that no Saa immunoreactivity was present from any of the groups (Fig. 3A1,A2, Table 1). However, the brains from the transgenic mice in the first group contained small, rare punctate-immunoreactive Saa protein in the cortex (Fig. 3A3). The deposits appeared as small amorphous structures similar to amyloid deposits, suggesting that acute-phase response induced AA amyloid deposition and that expression of the Saa precursor protein was absolutely required (Fig. 3A3, Table 1). In the second group of the transgenic mice, more and larger Saa immunoreactivity was detected in cortex (Fig. 3A4) and hippocampus (data not shown). Immunohistochemical analysis with anti-Saa immunostaining (Fig. 3B1) and thioflavin-S staining (Fig. 3B2) demonstrated that these entities were indeed amyloidogenic in nature and specifically AA amyloid deposits. Amyloid associated proteins such as apoE and serum amyloid P component were also present in most deposits (data not shown).



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  		Figure 3.   LPS induced AA amyloid deposition in SYNI-Saa1 transgenic mouse brain. A, Coronal sections of the neocortex stained with anti-mouse Saa. A1, Eight-month-old nontransgenic mice were injected with LPS twice a week for 1 month. A2, Eight-month-old nontransgenic mice were injected with LPS twice a week for 1 month and were killed after 1 more month without further LPS injections. A3, Eight-month-old transgenic mice treated the same as A1. A4, Eight-month-old transgenic mice treated the same as A2. B, Coronal sections of the hippocampus from 8-month-old transgenic mice treated the same as A4 and stained with rabbit anti-mouse Saa antibody (B1). Serum amyloid A deposits were also reactive with thioflavin-S (B2, serial sections). C, Coronal sections as in B, showing vascular amyloid deposits in the brain on transgenic mice stained with anti-SAA antibodies (C1) or thioflavin-S (C2). D, Colocalization of Saa immunoreactivity (rhodamine-conjugated secondary antibody) and thioflavin-S staining in 18-month-old transgenic mice. Sections were stained with thioflavin-S (D2) followed by anti-mouse Saa antibodies and rhodamine-conjugated secondary antibody (D1). D3 is the merged image of D1 and D2. Arrows indicate thioflavin-S-negative deposits. Representative samples of N = 8 per group. E, RNA and protein expression in Saa transgenic mice. Northern blot of RNA isolated from Saa transgenic mouse brains (E1). CON, Control transgenic mice; LPS, LPS-injected transgenic mice. E2, Plasma Saa levels in transgenic mice minus and plus LPS. E3, Saa protein levels in the brain of nontransgenic and transgenic mice. C, Control; L, LPS-treated animals.


                              
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  		Table 1.   AA amyloid deposition in transgenic mice brain

Vascular amyloid deposition in the SYNI-Saa1 transgenic mouse

The presence of vascular amyloid in AD has led to several hypotheses regarding the origin of the fibrillar material. The first suggests that the deposits result from circulating plasma levels of Abeta that eventually deposit in the vasculature (Poduslo et al., 1997; Mackic et al., 1998). The second hypothesis proposes that the Abeta peptide is derived from the brain and neurons that surround the vessels in the brain (Calhoun et al., 1999; Van Dorpe et al., 2000). The final source of vascular amyloid could be the cells of the vascular wall, i.e., endothelial cells, smooth muscle cells, or possibly macrophages (Natte et al., 1999). Figure 3C shows the presence of AA amyloid in the cerebrovasculature of inflammation-induced transgenic mice both by immunostaining (Fig. 3C1) and by thioflavin-S staining (Fig. 3C2). No amyloid is detected in nontransgenic mice in the absence or presence of inflammation (data not shown). Transgenic mice do not have detectable levels of Saa1 in the plasma (compared with nontransgenic mice), suggesting that the AA amyloid present in the vasculature originates from neuronal expression of the transgene. Examination of the amyloid deposits indicates that in both the Saa-immunoreactive section and the thioflavin-S-positive section, the amyloid extends into the neuronal region, suggesting that the development of the vascular lesion may have a neuronal origin.

Age-dependent increase in amyloid formation

When 18-month-old transgenic mice were subjected to the inflammatory protocol, they showed a faster, more robust and greater deposition of amyloid in the brain than the 8-month-old animals. This trend was confirmed by double staining with thioflavin-S and anti-mouse Saa antibodies (Fig. 3, compare D, B, respectively, and Table 2). The older animals showed an increase in both the number and size of deposits in the brain. Most Saa-positive and thioflavin-S-positive staining colocalized, as demonstrated in Figure 3D3. However, several small Saa-positive deposits did not have corresponding thioflavin-S staining (Fig. 3, compare arrows in D1, D2). This lack may represent the preamyloid state in which the precursors aggregate before conversion to amyloid fibrils. These results suggest that aging plays an important role in the pathogenesis of the disease.


                              
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  		Table 2.   Age and inflammation-dependent AA amyloid deposition

To determine the mechanism of increased amyloid formation in the aged mice, we measured mRNA and protein levels of Saa in the transgenic mice (Fig. 3E). Brain tissue from transgenic mice revealed no increase in mRNA expression of the Saa gene in either young mice or old mice in the absence or presence of inflammatory response (Fig. 3E1). However, mRNA levels in the liver are dramatically increased in LPS-injected animals (data not shown), and the plasma Saa protein increased accordingly (Fig. 3E2). No detectable Saa was found in the brains of nontransgenic mice, in the presence or absence of acute-phase inflammation (LPS injection). In contrast, the level of protein in the brain was significantly increased the transgenic mice but did not showed a significant rise in protein levels 24 hr after inflammation (data not shown). Two months after beginning the injections of LPS, there was a significant increase in Saa protein in the brain (Fig. 3E3), and there was a much larger increase of Saa protein in older transgenic mice. However, CSF levels of Saa protein did not significantly change (data not shown). These data suggest that the increase in Saa protein in the brain is the result of accumulation of the Saa protein into amyloid because of the failure of distinguishable changes in transcriptional regulation. Therefore, it is unlikely that the amyloid is caused by altered Saa levels in the brain and is more likely the result of activation of microglial cells and subsequent deposition of Saa into amyloid.

Inflammatory response in brains of SYNI-Saa1 transgenic mice

To examine the effects of LPS action on the deposition of AA amyloid in our transgenic model, we measured cytokine levels 24 hr after systemic LPS injections (Fig. 4). Wild-type and transgenic animals had low but detectable levels of IL-1beta , IL-6, and TNF-alpha in the brain before LPS injection (Fig. 4A). After LPS stimulation, cytokine mRNA (Fig. 4A) and protein (Fig. 4B) levels increased approximately threefold in the brains of both transgenic and nontransgenic animals, indicating that the LPS was capable of inducing an inflammatory response (Fishkin and Winslow, 1997). These changes in the levels of cytokines were transient and returned to control values within 48-72 hr (data not shown). Repeated injections of LPS continually induced cytokine expression in the brain (data not shown). These changes are similar to those seen in systemic amyloidosis where injection of LPS or casein induces inflammation but the Saa proteins form amyloid deposits only under chronic inflammatory conditions (Axelrad et al., 1982). Additionally, when 8-month-old and 18-month-old brain IL-6 levels were compared after LPS injection, the older animals showed a larger increase in IL-6 levels, indicating a more robust proinflammatory cytokine response (Fig. 4C). These increased levels of cytokines in aged animals may account for some of the differences seen in amyloid deposition (Fig. 3, Table 2).



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  		Figure 4.   Analysis of cytokine expression in the brain of transgenic and nontransgenic mice. A, RT-PCR analysis of cytokine gene expression in mice. Eight-month-old SYNI-Saa1 transgenic mice and nontransgenic mice were subjected to LPS injections. After 24 hr, brains from control and LPS-treated animals were removed, and total RNA was isolated for RT-PCR. Levels of IL-6 (black bars), IL-1beta (hatched bars), and TNF-alpha (gray bars) were given in arbitrary units representing the ratios between cytokine mRNA and GAPDH mRNA levels. *p < 0.01, compared with wild-type control; **p < 0.005, compared with transgenic control. B, Analysis of cytokine levels in mice. Eight-month-old SYNI-Saa1 transgenic and nontransgenic mice were subjected to LPS as described above. After 24 hr, brains were removed, and total proteins were isolated for ELISA. Levels of IL-6 (black bars, in units per milliliter), IL-1beta (hatched bars, in units per milliliter), and TNF-alpha (open bars, in picograms per milliliter) were determined. *p < 0.005, compared with wild-type control; **p < 0.001, compared with transgenic control. N = 8 mice per group. C, Cytokine levels in 8-month-old and 18-month-old mice. At the indicated ages, mice were injected with LPS and after 24 hr, IL-6 levels were determined in the brain. *p < 0.005, compared with 8-month-old LPS-injected animals.

Further examination of brains from animals treated with LPS showed the presence of activated astrocytes in brain areas with AA amyloid deposition. Figure 5 illustrates the immunostaining seen in transgenic and nontransgenic mice in the presence and absence of inflammation. Immunoreactive staining of GFAP, which detects activated astrocytes, was present in the transgenic mice subjected to the inflammatory protocol (Fig. 5A1). When compared with transgenic mice in the absence of inflammation (Fig. 5A3) or the nontransgenic mice with or without inflammation (Fig. 5A2,A4, respectively), these animals showed a dramatic increase in GFAP staining and astrocyte activation. Figure 5B shows that the GFAP-positive activated astrocytes (Fig. 5B2) were present in the areas surrounding the amyloid deposits (Fig. 5B1). Staining of serial sections showed that the activated astrocytes were localized to the region surrounding the amyloid deposit. In transgenic mice, cytokine levels were increased in the presence of amyloid deposits and activated astrocytes as determined by increased IL-6 mRNA expression (Fig. 5C). Immunostaining with anti-phosphotyrosine antibodies detected the presence of activated microglial cells surrounding the amyloid deposits (data not shown). These results indicate that the amyloid deposits can further enhance inflammation in the brain.



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  		Figure 5.   Association of activated astrocytes with AA amyloid deposits. A, Immunohistochemical analysis of brains from transgenic and nontransgenic mice in the presence and absence of LPS. A1, SYNI/Saa1 transgenic mouse injected with LPS (1 month injection with LPS, 1 month no injection); A2, nontransgenic mouse injected with LPS; A3, transgenic mouse with no injection; A4, nontransgenic mouse with no injection. Tissue was stained with rabbit anti-mouse GFAP. Representative sample of N = 6 mice per group. B, Activated astrocytes are associated with the amyloid deposits. B1, Immunohistochemical analysis using an anti-Saa antibody; B2, serial section immunostained with the anti-GFAP antibody, showing colocalization of the antibodies. Higher magnification of A1. C, Plot of IL-6 expression in the brain of transgenic and nontransgenic animals. Mice were prepared as described above, 1 month with LPS and 1 month without LPS, and the brains analyzed for IL-6 expression. *p < 0.005; N = 7 per group.

Inhibition of inflammation reduces amyloid deposition in the Saa1 transgenic mice

To determine the effect of inflammation on amyloid deposition in the brain of Saa1 transgenic animals, 8-month-old animals were injected with indomethacin (30 mg · kg-1 · d-1, i.p.) during and after LPS injections and examined for amyloid deposition and cytokine expression (Fig. 6). Animals injected with saline (Fig. 6A1) showed the typical amyloid deposition seen in transgenic mice after LPS stimulation. Injection of the NSAID indomethacin dramatically reduced the number and size of amyloid deposits seen in the Saa1 mice (Fig. 6A2, Table 2). At the same time, cytokine levels were significantly decreased in the transgenic mice treated with indomethacin compared with control transgenic mice (Fig. 6B). These results support a role for inflammation in the development of amyloid deposition in the brain of the transgenic mice and indicate that this process may be present in the brains of patients with AD and that therapeutic intervention with NSAIDS may provide substantial inhibition of the disease.



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  		Figure 6.   Indomethacin reduces amyloid deposition and cytokine expression in transgenic mice. A, Eight-month-old transgenic Saa1 mice (n = 12) were injected with LPS as described in Figure 5, and half (n = 6) were injected with indomethacin (30 mg/kg) daily for the entire period. Animals were killed and examined for amyloid deposition. Tissues were stained with anti-Saa antibody. A1, Transgenic with LPS only; A2, transgenic with LPS plus indomethacin. B, IL-6 mRNA expression in transgenic mice injected with indomethacin. Mice were prepared as in A, and mRNA analyzed for IL-6 expression (ratio of IL-6 to GAPDH mRNA). *p < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several transgenic mouse models have been generated in the attempts of reproducing many of the pathological hallmarks of AD (Games et al., 1995; Hsiao et al., 1996; Sturchler-Pierrat et al., 1997). In these models, overexpression of mutant APP proteins results in increased levels of Abeta peptides in the brains of the transgenic animals. Amyloid plaques, which are an important pathological feature of AD, have been demonstrated in these mice along with other aspects such as astrogliosis and microgliosis (Bornemann et al., 2001). However, results from these transgenic models have been unable to explain if inflammation is actively involved in amyloidogenic process or just a secondary event in AD (Lim et al., 2000). A number of acute phase proteins are associated with amyloid plaques, and it is hypothesized that acute phase response plays important roles in the pathogenesis of AD.

Here, we have generated transgenic mice for a brain amyloid model by expressing the mouse Saa1 protein using the neuronal specific synapsin promoter. Saa1 is the amyloidogenic form of the mouse Saa proteins; however, it is not normally expressed in the mouse brain. One advantage of this model is that the protein expressed is murine, not from another species. In mutant APP transgenic models, overexpression of a human protein may cause unexpected results. In amyloid beta  precursor protein (Abeta PP) transgenic mice, Abeta amyloid plaques are formed without intervention (Games et al., 1995; Hsiao et al., 1996; Hsia et al., 1999), and we hypothesize that the overexpression of human APP and resultant high concentration of Abeta in mouse brain causes focal inflammation, which in turn facilitates Abeta deposition. Expression of Saa1 alone is not sufficient to allow for amyloid deposition (Games et al., 1995). However, when a systemic acute phase response is induced, the SYNI-Saa1 transgenic mice developed amyloid deposits in the brain. These results suggest that inflammation contributes to the process of amyloid formation and that systemic inflammation can increase cytokine expression in the brain and initiate or enhance amyloid deposition via activation of microglial cells and/or amyloid associated proteins.

The Abeta peptide itself can induce a local inflammatory response (Cotman et al., 1996; Bradt et al., 1998; Galimberti et al., 1999; Paris et al., 2000). This local inflammatory reaction may help to initiate and propagate the amyloidogenic process, establishing a vicious cycle in which amyloid deposits further activate microglia and astrocytes that stimulate more cytokine production such as IL-1beta , IL-6, and TNF-alpha , which in turn causes more amyloid deposition (Fig. 5). Treatment with anti-inflammatory drugs, such as indomethacin and ibuprofen, may help to reduce amyloid deposition and eventually prevent cellular degeneration and improve memory (our data and Lim et al., 2000). Our finding suggests that factors such as acute infection, head trauma, or stroke can trigger inflammatory process in the brain, which may play a crucial role in the development of the disease.

One of the most interesting findings of our in vivo data concerns the changes in amyloid deposition as the age of the mice increased. Previous studies in the APP transgenic mice have shown that over time Abeta deposits increased. This finding suggested that either the accumulation of Abeta peptide eventually leads to the formation of amyloid fibrils or the susceptibility of these animals to amyloid deposition is related to age (Games et al., 1995; Hsiao et al., 1996; Hsia et al., 1999). Deciphering the potential role each of these factors plays in the formation of the amyloid deposits is difficult because of the intractable nature of the Abeta peptide and its rapid ability to form amyloid fibrils. Our model, because it does not form Saa deposits unless inflammation is induced, allowed us to test the hypothesis that age increases the susceptibility of the brain to amyloid deposition. Eight-month-old Saa1 transgenic mice developed no AA amyloid deposits after 1 month of LPS treatment and a subsequent month without LPS (Fig. 3A). However, 18-month-old animals with the same inflammatory paradigm developed more and larger deposits than did the 8-month-old animals. These data suggest that both the presence of an amyloidogenic protein is required and that the progression of age contributes to the pathogenesis of the disease.

Another striking finding was the presence of cerebral amyloid angiopathy (CAA) in the Saa1 transgenic mice. In Abeta -derived CAA, the leptomeningeal and cortical vessels are affected, and the deposition is associated with degeneration of smooth muscle cells, endothelial cells, and pericytes. Excessive deposition of Abeta in the vasculature can result in cerebrovascular hemorrhage and dramatically increases the risk for stroke (Winkler et al., 2001). Cerebrovascular deposition of amyloid is a common occurrence in individuals with AD (Rosand et al., 2000). However, the origin and mechanism of the pathological finding are still unknown. It is hypothesized that the Abeta peptide (or other amyloidogenic protein) originates from the blood, the vessel wall, or from neurons through drainage with the interstitial fluid around the vessels (Burgermeister et al., 2000). The data presented here help to strengthen the argument that deposition of amyloid into the cerebrovasculature derives from neurons. Our mice express the transgenic Saa1 gene only in neurons, yet they develop cerebral amyloid angiopathy in the presence of a systemic acute phase stimulus. In the absence of the transgene, systemic inflammation results in elevation of the endogenous Saa genes, but there is no detectable CAA in these animals. Under noninflammatory conditions, the level of Saa protein in the plasma of SYNI-Saa1 transgenic mice does not differ from wild-type mice, suggesting that increased plasma levels of Saa do not directly contribute to the deposition of amyloid in the vasculature. It is possible that the combination of plasma and neuron-derived Saa expression may contribute to the deposition of AA amyloid in the vessels. The development of an Saa-deficient mouse would help to define the specific involvement of neuronal or plasma Saa in the formation of CAA.

In conclusion, the generation of this mouse model has helped to elucidate several mechanisms that may be important in the development of AD. Our data show that inflammation plays a crucial role in the development of Saa1 amyloid deposits in the brain. Our data further show that the role played by aging in incidence and progression of AD also is important in that aged mice developed more aggressive and more involved amyloid deposition. Finally, the mechanisms necessary for the deposition of amyloid in the vasculature have been elusive and the data here suggest that the neuron-derived amyloidogenic precursor is capable of forming vascular amyloid. This model will be important in helping to understand the relative role of inflammation and other factors that will further the development of improved therapies for preventing AD.


    FOOTNOTES

Received Aug. 27, 2001; revised Feb 14, 2002; accepted April 23, 2002.

This work was supported by United States Public Health Service Grants NS31220, AG12891, and NS39588 and the Stroke Program of the Sanders-Brown Center on Aging.

Correspondence should be addressed to Dr. Mark S. Kindy, Department of Physiology and Neuroscience, Medical University of South Carolina, 650 MUSC Complex, Suite 403, Charleston, SC 29425. E-mail: mskindy{at}musc.edu.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

  • Abraham CR, Slekoe DJ, Potter H (1988) Immunochemical identification of the serine protease inhibitor alpha 1-antichymotrypin in the brain amyloid deposits of Alzheimer's disease. Cell 52:487-501[ISI][Medline].
  • Axelrad MA, Kisilevsky R, Willmer J, Chen SJ, Skinner M (1982) Further characterization of amyloid-enhancing factor. Lab Invest 47:139-146[ISI][Medline].
  • Bauer J, Strauss S, Schreiter-Gasser U, Ganter U, Schlegel P, Witt I, Yolk B, Berger M (1991) Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices. FEBS Lett 285:111-114[ISI][Medline].
  • Bornemann KD, Wiederhold KH, Pauli C, Ermini F, Stalder M, Schnell L, Sommer B, Jucker M, Staufenbiel M (2001) Abeta-induced inflammatory processes in microglia cells of APP23 transgenic mice. Am J Pathol 158:63-73[Abstract/Free Full Text].
  • Bradt BM, Kolb WP, Cooper NR (1998) Complement-dependent proinflammatory properties of the Alzheimer's disease beta-peptide. J Exp Med 188:431-438[Abstract/Free Full Text].
  • Breitner JC (1996) The role of anti-inflammatory drugs in the prevention and treatment of Alzheimer's disease. Annu Rev Med 47:401-411[ISI][Medline].
  • Burgermeister P, Calhoun ME, Winkler DT, Jucker M (2000) Mechanisms of cerebrovascular amyloid deposition. Lessons from mouse models. Ann NY Acad Sci 903:307-316[Abstract/Free Full Text].
  • Calhoun ME, Burgermeister P, Phinney AL, Stadler M, Tolnay M, Wiederhold K-H, Abramowski D, Sturchler-Pierrat C, Sommer B, Staufenbiel M, Jucker M (1999) Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid. Proc Natl Acad Sci USA 96:14088-14093[Abstract/Free Full Text].
  • Cotman CW, Tenner AJ, Cummings BJ (1996) beta -amyloid converts an acute phase injury response to chronic injury responses. Neurobiol Aging 17:723-731[ISI][Medline].
  • Eikelenboom P, Stam FC (1982) Immunoglobulins and complement factors in senile plaques. An immunoperoxidase study. Acta Neuropathol (Berl) 57:239-242[Medline].
  • Fishkin RJ, Winslow JT (1997) Endotoxin-induced reduction of social investigation by mice: interaction with amphetamine and anti-inflammatory drugs. Psychopharmacology 132:335-341[Medline].
  • Friman C, Pettersson T (1996) Amyloidosis. Curr Opin Rheumatol 8:62-71[Medline].
  • Gahtan E, Overmier JB (1999) Inflammatory pathogenesis in Alzheimer's disease: biological mechanisms and cognitive sequelae. Neurosci Biobehav Rev 23:615-633[Medline].
  • Galimberti D, Baron P, Meda L, Prat E, Scarpini E, Delgado R, Catania A, Lipton JM, Scarlato G (1999) Alpha-MSH peptides inhibit production of nitric oxide and tumor necrosis factor-alpha by microglial cells activated with beta-amyloid and interferon gamma. Biochem Biophys Res Commun 263:251-256[ISI][Medline].
  • Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F, Duido T, Hagopian S, Johnson-Wood K, Khan K, Lee M, Leibowitz P, Little S, Masliah E, McConlogue L, Montoya-Zavala M, Mucke L, Paganini L, Penniman E, Power M, Schenk D, Seubert P, Snyder B, Soriano F, Tan H, Vitale J, Wadsworth S, Wolozin B, Zhao J (1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta -amyloid precursor protein. Nature 373:523-527[Medline].
  • Ganowiak K, Hultman P, Engstrom U, Gustavsson A, Westermark P (1994) Fibrils from synthetic amyloid-related peptides enhance development of experimental AA amyloidosis in mice. Biochem Biophys Res Commun 199:306-312[ISI][Medline].
  • Gatti S, Bartfai T (1993) Induction of tumor necrosis factor-alpha mRNA in the brain after peripheral endotoxin treatment: comparison with interleukin-1 family and interleukin-6. Brain Res 624:291-294[ISI][Medline].
  • Griffin WS, Stanley LC, Ling C, White L, Macleod V, Perrot LJ, White CL, Arora C (1989) Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc Natl Acad Sci USA 86:7611-7615[Abstract/Free Full Text].
  • Higgins LS, Holtzman DM, Rabin J, Mobley WC, Cordell B (1994) Transgenic mouse brain histopathology resembles early Alzheimer's disease. Ann Neurol 35:598-607[ISI][Medline].
  • Hoffman JS, Ericsson LH, Eriksen N (1984) Murine tissue amyloid protein AA. J Exp Med 159:641-646[Abstract/Free Full Text].
  • Hogan B, Costantini F, Lacy E (1982) In: Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  • Howland DS, Hemmendinger JS, Carroll PD, Estes PS, Melloni Jr RH, DeGennaro LJ (1991) Positive- and negative-acting promoter sequences regulate cell type-specific expression of the rat synapsin I gene. Mol Brain Res 11:345-353[Medline].
  • Hsia AY, Masliah E, McConlogue L, Yu GQ, Tatsuno G, Hu K, Kholodenko D, Malenka RC, Nicoll RA, Mucke L (1999) Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc Natl Acad Sci USA 96:3228-3233[Abstract/Free Full Text].
  • Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G (1996) Correlative memory deficits, Abeta elevation and amyloid plaques in transgenic mice. Science 274:177-178[ISI][Medline].
  • Kindy MS, de Beer FC (1999) A mouse model for serum amyloid A amyloidosis. Methods Enzymol 309:701-716[Medline].
  • Kindy MS, Rader DJ (1998) Reduction in amyloid A amyloid formation in apolipoprotein-E-deficient mice. Am J Pathol 152:1387-1395[Abstract].
  • Kindy MS, McCormack JE, Buckler AJ, Levine RA, Sonenshien GE (1987) Independent regulation of transcription of the two strands of the c-myc gene. Mol Cell Biol 7:2857-2862[Abstract/Free Full Text].
  • Kindy MS, King AR, Perry G, deBeer MC, deBeer FC (1995) Association of apolipoprotein E with murine amyloid A protein amyloid. Lab Invest 73:469-475[ISI][Medline].
  • Kindy MS, Yu J, Guo J, Zhu H (1999) Apolipoprotein serum amyloid A in Alzheimer's disease. J Alz Dis 1:155-167.
  • Liang J, Sloane JA, Wells JM, Abraham CR, Fine RE, Sipe JD (1997) Evidence for local production of acute phase response apolipoprotein serum amyloid A in Alzheimer's disease brain. Neurosci Lett 225:73-76[ISI][Medline].
  • Lim GP, Yang F, Chen P, Beech W, Teter B, Tran T, Ubeda O, Hsiao-Ashe K, Frautschy SA, Cole GM (2000) Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer's disease. J Neurosci 20:5709-5714[Abstract/Free Full Text].
  • Mackic JB, Weiss MH, Miao W, Kirkman E, Ghiso J, Calero M, Bading J, Frangione B, Zlokovic BV (1998) Cerebrovascular accumulation and increased blood-brain barrier permeability to circulating Alzheimer's amyloid beta peptide in aged squirrel monkey with cerebral amyloid angiopathy. J Neurochem 70:210-215[ISI][Medline].
  • McAdam KP, Sipe JD (1976) Murine model for human secondary amyloidosis: genetic variability of the acute-phase serum protein SAA response to endotoxins and casein. J Exp Med 144:1121-1127[Abstract/Free Full Text].
  • McGeer PL, McGeer EG (1998) Mechanisms of cell death in Alzheimer's disease: immunopathology. J Neural Transm Suppl 54:1131-1137.
  • McGeer PL, Schulzer M, McGeer EG (1995) Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer's disease: a review of 17 epidemiologic studies. Neurology 95:425-432.
  • Meek RL, Benditt EP (1986) Amyloid a gene family expression in different mouse tissues. J Exp Med 164:2006-2017[Abstract/Free Full Text].
  • Meek RL, Hoffman JS, Benditt EP (1986) Amyloidogenesis: one serum amyloid A isotype is selectively removed from the circulation. J Exp Med 163:499-510[Abstract/Free Full Text].
  • Natte R, de Boer WI, Maat-Schieman ML, Baelde HJ, Vinters HV, Roos RA, van Duinen SG (1999) Amyloid beta precursor protein-mRNA is expressed throughout cerebral vessel walls. Brain Res 828:179-183[Medline].
  • Paris D, Town T, Mori T, Parker TA, Humphrey J, Mullan M (2000) Soluble beta-amyloid peptides mediate vasoactivity via activation of a pro-inflammatory pathway. Neurobiol Aging 21:183-197[ISI][Medline].
  • Poduslo JF, Curran GL, Haggard JJ, Biere AL, Selkoe DJ (1997) Permeability and residual plasma volume of human, Dutch variant, and rat amyloid beta-protein 1-40 at the blood brain barrier. Neurobiol Dis 4:27-34[Medline].
  • Rogers J, Cooper NR, Webster S, Schultz J, McGeer PL, Styren SD, Civin WH, Brachova L, Bradt B, Ward P, Lieberburg I (1992) Complement activation by beta -amyloid in Alzheimer's disease. Proc Natl Acad Sci USA 92:10016-10020.
  • Rosand J, Hylek EM, O'Donnell HC, Greenberg SM (2000) Warfarin-associated hemorrhage and cerebral amyloid angiopathy: a genetic and pathologic study. Neurology 55:947-951[Abstract/Free Full Text].
  • Selkoe D (1994) Normal and abnormal biology of the beta-amyloid precursor protein. Annu Rev Neurosci 17:489-517[ISI][Medline].
  • Selkoe DJ (1999) Translating cell biology into therapeutic advances in Alzheimer's disease. Nature 399:A23-A31[Medline].
  • Shiroo M, Kawahara E, Nakanishi I, Migita S (1987) Specific deposition of serum amyloid A protein 2 in the mouse. Scand J Immunol 26:709-716[Medline].
  • Sipe JD (1992) Amyloidosis. Annu Rev Biochem 61:947-975[ISI][Medline].
  • Smith MA, Hirai K, Hsiao K, Pappolla MA, Harris PL, Siedlak SL, Tabaton M, Perry G (1998) Amyloid-beta deposition in Alzheimer's transgenic mice is associated with oxidative stress. J Neurochem 70:2212-2215[ISI][Medline].
  • Smith MA, Rottkamp CA, Numomura A, Raina AK, Perry G (2000) Oxidative stress in Alzheimer's disease. Biochim Biophys Acta 1502:139-144[Medline].
  • Smith MA, Taneda S, Richey PL, Miyata S, Yan SD, Stern D, Sayre LM, Monnier VM, Perry G (1994) Advanced Maillard reaction end products are associated with Alzheimer's disease pathology. Proc Natl Acad Sci USA 92:2016-2020.
  • Sturchler-Pierrat C, Abramowski D, Duke M, Wiederhold K, Mistl C, Rothacher S, Ledermann B, Burki K, Frey P, Paganetti PA, Waridel C, Calhoun ME, Jucker M, Probst A, Staufenbiel M, Sommer B (1997) Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc Natl Acad Sci USA 94:13287-13292[Abstract/Free Full Text].
  • Tehranian R, Hasanvan H, Iverfeldt K, Post C, Schultzberg M (2001) Early induction of interleukin-6 mRNA in the hippocampus and cortex of APPsw transgenic mice Tg2576. Neurosci Lett 301:54-58[Medline].
  • Vandenabeele P, Fiers W (1991) Is amyloidogenesis during Alzheimer's disease due to an IL-1/IL-6 mediated "acute phase response" in the brain? Immunol Today 12:217-219[ISI][Medline].
  • Van Dorpe J, Smeijers L, Dewachter I, Nuyens D, Spittaels K, Van Den Haute C, Mercken M, Moechars D, Laenen I, Kuiperi C, Bruynseels K, Tesseur I, Loos R, Vanderstichele H, Checler F, Sciot R, Van Leuven F (2000) Prominent cerebral amyloid angiopathy in transgenic mice overexpressing the London mutant of human APP in neurons. Am J Pathol 157:1283-1298[Abstract/Free Full Text].
  • Westermark GT, Engstrom U, Westermark P (1992) The N-terminal segment of protein AA determines its fibrillogenic property. Biochem Biophys Res Com 182:27-33[ISI][Medline].
  • Winkler DT, Bondolfi L, Herzig MC, Jann L, Calhoun ME, Wiederhold KH, Tolnay M, Staufenbiel M, Jucker M (2001) Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy. J Neurosci 21:1619-1627[Abstract/Free Full Text].
  • Yamamoto K, Shiroo M, Migita S (1986) Diverse gene expression for isotypes of murine serum amyloid A protein during acute phase reaction. Science 232:227-229[Abstract/Free Full Text].
  • Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, Slattery T, Zhao L, Nagashima M, Morser J, Migheli A, Nawroth P, Stern D, Schmidt AM (1996) RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature 382:685-691[Medline].
  • Yankner BA (1996) Mechanisms of neuronal degeneration in Alzheimer's disease. Neuron 16:921-932[ISI][Medline].

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