Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease

Abstract

The accumulation of amyloid beta protein (Abeta) in the Tg2576 mouse model of Alzheimer's disease (AD) was evaluated by ELISA, immunoblotting, and immunocytochemistry. Changes in Abeta begin at 6-7 months as SDS-insoluble forms of Abeta42 and Abeta40 that require formic acid for solubilization appear. From 6 to 10 months, these insoluble forms increase exponentially. As insoluble Abeta appears, SDS-soluble Abeta decreases slightly, suggesting that it may be converting to an insoluble form. Our data indicate that it is full-length unmodified Abeta that accumulates initially in Tg2576 brain. SDS-resistant Abeta oligomers and most Abeta species that are N-terminally truncated or modified develop only in older Tg2576 mice, in which they are present at levels far lower than in human AD brain. Between 6 and 10 months, when SDS-insoluble Abeta42 and Abeta40 are easily detected in every animal, histopathology is minimal because only isolated Abeta cores can be identified. By 12 months, diffuse plaques are evident. From 12 to 23 months, diffuse plaques, neuritic plaques with amyloid cores, and biochemically extracted Abeta42 and Abeta40 increase to levels like those observed in AD brains. Coincident with the marked deposition of Abeta in brain, there is a decrease in CSF Abeta and a substantial, highly significant decrease in plasma Abeta. If a similar decline occurs in human plasma, it is possible that measurement of plasma Abeta may be useful as a premorbid biomarker for AD.

Fig. 1.

Expression of βAPP, CTF, and Aβ in brain and systemic organs of Tg2576 and nontransgenic mice. Immunoblots in A–F were labeled with anti-C (Saeko) (A, C, E), which detects both human and mouse βAPP, or with 6E10 (B,D, F), which specifically detects human βAPP. A, B, Immunoblots of βAPP and CTF in transgenic and nontransgenic mice of various ages (months). Immunoblots were prepared from 16% Tricine gels. C–F, Immunoblots of SDS extracts from systemic organs of a 10.9 month Tg2576 mouse (C, D) and a 9.3 month nontransgenic mouse (E, F). Immunoblots were prepared from 10–20% Tricine gels loaded at 20 mg/lane total protein. Tg, Transgenic;NTg, nontransgenic; Br, brain;H, heart; Lg, lung; Lv, liver; K, kidney; P, pancreas;Sp, spleen; St, stomach;Si, small intestine; Li, large intestine;M, muscle; Bo, bone; Sk, skin. G, Immunoblot of Aβ in SDS and FA extracts of systemic organs. The Aβ in SDS extracts (40 μl) was analyzed by 4G8 immunoprecipitation followed by immunoblotting with 4G8; FA acid extracts (40 μl), dried and resuspended, were also analyzed by immunoblotting with 4G8. H, Total Aβ (Aβ42 plus Aβ40) in SDS and FA extracts of systemic organs. Aβ42 and Aβ40 were analyzed by 3160/BC-05 and 3160/BA-27 ELISAs, respectively.

Fig. 2.

Immunohistochemistry of AD (A,B) and aging Tg2576 (C–P) brains. Serial sections of temporal cortex from AD brain and Tg2576 brains were labeled with BA-27, which is specific for Aβ40, or BC-05, which is specific for Aβ42. The age (months) of the Tg2576 brains is shown above the serial sections stained with BA-27 (top panel) and BC-05 (bottom panel). Aβ40 (stained by BA-27) and Aβ42 (stained by BC-05) are detected as dense microdeposits from 8 (M, N) to 12 (I, J) months. From 15 to 23 months (C–H), Aβ deposits increase in number and size and are detected both as cored plaques labeled by both BC-05 and BA-27 and as diffuse plaques, which are selectively labeled by BC-05. Sections are 5-μm-thick. Scale bar, 15 μm.

Fig. 3.

Aβ in aging Tg2576 brain. Aβ42 (A, C) and Aβ40 (B,D) were analyzed in Tg2576 brains sequentially extracted in 2% SDS (A, B) and 70% formic acid (C, D). The ELISA assay was 3160/BC05 for Aβ42 and 3160/BA27 for Aβ40. Note that the y-axes are logarithmic and that there was no detectable Aβ40 or Aβ42 in the formic acid extract of young (2–5 months) Tg2576 mice.

Fig. 4.

Analysis by immunoblotting of early Aβ deposition in Tg2576 brain. Aβ in the SDS (A,C) and formic acid (B, D) extracts from 4–10 month Tg2576 brains was analyzed on immunoblots labeled with BAN-50 (anti-Aβ1–16) (A,B) and 4G8 (anti-Aβ17–24) (C,D). Proteins were separated on 10–20% Tricine gels, and each lane shows the Aβ in 40 μl of the formic acid or SDS extract as described in Materials and Methods. Immunoblotting was first performed with BAN-50. The blots were then stripped and reblotted with 4G8. Note that SDS-soluble Aβ decreases transiently at 8 months, when SDS-resistant, formic acid-soluble Aβ appears. The arrows identify CTFβ, and thearrowheads identify Aβ.

Fig. 5.

Specific forms of Aβ in Tg2576 and AD brains.A–D, Immunoblot analysis of SDS (A,C) and formic acid (B, D) extracts of 21M Tg2576 mouse brains and AD brains labeled with BAN-50 (A, B) or 4G8 (C,D). Two microliters of the SDS or FA extract (dried and resuspended) were directly added to each lane; proteins were separated on 10–20% Tricine gels. E,F, Immunoblot analysis of SDS (E) and formic acid (F) extracts of AD and 23M Tg2576 brains labeled with the following: 4G8, which detects both N-terminally modified and unmodified Aβ; anti-AβN1(D), which detects the unmodified N terminus; anti-N1(iD), which recognizes isomerized forms (l-iso-Asp) of AβN1; anti-N1(rD), which detects stereoisomerized forms (rectus Asp) of AβN1; anti-AβN3(pE), which detects forms beginning with pyroglutamate at position 3; or anti-AβN11(pE), which recognizes forms beginning with pyroglutamate at position 11. The Aβ in 10 μl of SDS or FA extracts was examined on each lane. In the SDS extracts, Aβ was immunoprecipitated with the indicated antibody as described in Materials and Methods before separation and immunoblotting with the same antibody. In the FA extracts, Aβ was dried and resuspended as described in Materials and Methods before separation and immunoblotting. Proteins were separated on 16% Tricine gels.G–L, Time course of accumulation of formic acid-soluble Aβ in Tg2576 brains (8M–23M) and AD brains labeled with 4G8 (G), anti-N1(D) (H), anti-N1(rD) (I), anti-N3(pE) (J), BA-27 (K), or BC-05 (L). The Aβ in 10 μl of formic acid extract was examined on each lane, and proteins were separated on 16% Tricine gels.

Fig. 6.

Immunohistochemical analysis of modified forms of Aβ in aging Tg2576 brain. Serial sections (5 μm) of Tg2576 cerebral cortex were labeled with anti-N1(D), anti-N1(iD), anti-N1(rD), anti-N3(pE), and 4G8. The age of the mouse brain analyzed is shown at the top of each set of serial sections. Scale bar, 17 μm.

Fig. 7.

CSF and plasma Aβ in Tg2576 mice decline as Aβ is deposited in the brain. Total brain Aβ42 (A) and Aβ40 (B) were assayed by 3160/BC05 or 3160/BA27 ELISAs, respectively; see also Figure 2 and Table 2. Tg2576 CSF and plasma Aβ42 (C, E) were assayed by BNT77/BC05 ELISA, and Tg2576 CSF and plasma Aβ40 (D, F) were assayed by BAN50/BA27 ELISA. Both nontransgenic (NTg) CSF and plasma Aβ42 (G, I) and Aβ40 (H, J) were assayed with BNT77 capture. The number of Tg2576 CSF samples assayed for the four time groups are 9, 9, 16, and 11, totaling 45. The number of Tg2576 plasma samples assayed for the four time groups are 30, 18, 64, and 19, totaling 131. The decline for CSF Aβ42 is significant (p = 0.02), and the declines for plasma Aβ40 and Aβ42 are highly significant (Aβ42, p= 0.008; Aβ40, p = 0.006; Spearman's rank correlation for the 6–23 month age range). The number of nontransgenic CSF samples assayed for the three time groups are 2, 2, and 6, totaling 10. The number of nontransgenic plasma samples assayed for the four time groups are 7, 6, 12, and 6, totaling 31.