Inflammation-dependent cerebral deposition of serum amyloid a protein in a mouse model of amyloidosis

Abstract

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.

Fig. 1.

Saa1 transgenic construct and mice.A,Saa1 transgenic construct. A 4.5 kbBamHI fragment of the rat synapsin I promoter was linked to the 2.4 kb BglII–XhoI mouseSaa1 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 mouseSaa1 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, and7. One kilobase molecular weight markers are indicated (1 kb).

Fig. 2.

Saa transgene expression, tissue distribution, and protein levels. A, Northern blot analysis of brain RNA from transgenic and nontransgenic animals. The expression ofSaa1 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 ofSaa1 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.

Fig. 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 asA2. 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 andD2. 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.

Fig. 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-1β (hatched bars), and TNF-α (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-1β (hatched bars, in units per milliliter), and TNF-α (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.

Fig. 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 ofN = 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.

Fig. 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.