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. 2002 Aug 6;99(16):10837-42.
doi: 10.1073/pnas.162350199. Epub 2002 Jul 15.

Prominent Neurodegeneration and Increased Plaque Formation in Complement-Inhibited Alzheimer's Mice

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Free PMC article

Prominent Neurodegeneration and Increased Plaque Formation in Complement-Inhibited Alzheimer's Mice

Tony Wyss-Coray et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Abnormal accumulation of beta-amyloid (Abeta) in Alzheimer's disease (AD) is associated with prominent brain inflammation. Whereas earlier studies concluded that this inflammation is detrimental, more recent animal data suggest that at least some inflammatory processes may be beneficial and promote Abeta clearance. Consistent with these observations, overproduction of transforming growth factor (TGF)-beta1 resulted in a vigorous microglial activation that was accompanied by at least a 50% reduction in Abeta accumulation in human amyloid precursor protein (hAPP) transgenic mice. In a search for inflammatory mediators associated with this reduced pathology, we found that brain levels of C3, the central component of complement and a key inflammatory protein activated in AD, were markedly higher in hAPP/TGF-beta1 mice than in hAPP mice. To assess the importance of complement in the pathogenesis of AD-like disease in mice, we inhibited C3 activation by expressing soluble complement receptor-related protein y (sCrry), a complement inhibitor, in the brains of hAPP mice. Abeta deposition was 2- to 3-fold higher in 1-year-old hAPP/sCrry mice than in age-matched hAPP mice and was accompanied by a prominent accumulation of degenerating neurons. These results indicate that complement activation products can protect against Abeta-induced neurotoxicity and may reduce the accumulation or promote the clearance of amyloid and degenerating neurons. These findings provide evidence for a role of complement and innate immune responses in AD-like disease in mice and support the concept that certain inflammatory defense mechanisms in the brain may be beneficial in neurodegenerative disease.

Figures

Fig 1.
Fig 1.
Increased complement C3 expression in hAPP/TGF-β1 and TGF-β1 transgenic mice. (A) Brains from hAPP (white bars) and hAPP/TGF-β1 (black bars) mice at the indicated ages were divided sagitally, and relative C3 mRNA levels were measured by RNase protection assay in one hemibrain. (B) In the opposite hemibrain, average numbers of thioflavin S-positive plaques per 40-μm brain section (5–6 sections per mouse; hippocampus plus neocortex) were counted. Values are mean ± SEM from 4–6 mice per group. *, P < 0.05; **, P < 0.001 by t test. (C) Western blot analysis of solubilized total brain homogenates from 15-month-old hAPP, hAPP/TGF-β1, TGF-β1, and nontransgenic littermate mice was performed under nonreducing conditions with an anti-C3 (C3c) antibody. The α/β dimer of C3 (185 kDa) is indicated on the right.
Fig 2.
Fig 2.
Increased Aβ accumulation and amyloid formation in hAPP/sCrry mice. (AF) Brains from 10- to 12-month-old hAPP (n = 6) and hAPP/sCrry (n = 8) mice were dissected and analyzed for Aβ accumulation and amyloid formation. (A and B) Aβ immunostaining in the hippocampus and neocortex of an hAPP (A) and an hAPP/sCrry (B) mouse. (C) The area occupied by Aβ immunoreactivity was significantly larger in hAPP/sCrry than in hAPP mice. Values are mean ± SEM. **, P < 0.01 by t test. (D) Total Aβ (Aβ1-x, black + gray bars) and Aβ1–42 levels (gray bars) in neocortex and hippocampus of hAPP and hAPP/sCrry mice. Values are mean ± SEM. **, P = 0.028; *, P = 0.039 by t test. (E and F) Congo red staining in the hippocampus of an hAPP/sCrry mouse viewed with crosspolarized filters (E) or under normal light (F). (G) Aβ1–42/Aβ1-x ratios in neocortex and hippocampus of 3-month-old hAPP/sCrry (n = 9; black bars) and hAPP (n = 8; white bars) mice as measured by ELISA. Values are mean ± SEM. *, P < 0.05 by t test. [Scale bars: 250 μm (A and B), 100 μm (E and F).]
Fig 3.
Fig 3.
Transgene and complement expression in hAPP and hAPP/sCrry brains. (A) Similar levels of hAPP were detected in hippocampal homogenates from 3-month-old hAPP and hAPP/sCrry mice by Western blot analysis with the 8E5 antibody. APP, hAPP isoforms; asterisk indicates a nonspecific band. (B) Similar levels of C3 were detected in hippocampal homogenates from 3-month-old mice of four genotypes by Western blot analysis with an anti-C3 (C3c) antibody. (C) Relative C3d immunoreactivity (IR) in the stratum radiatum of the CA3 hippocampal region of 12-month-old mice calculated from four measurements per case. Values are mean ± SEM from three mice per genotype.
Fig 4.
Fig 4.
Prominent neurodegeneration and reduced microgliosis in hAPP/sCrry mice. Brain sections from 10- to 12-month-old mice from a cross of hAPP with sCrry mice were analyzed for neurodegeneration and microglial activation. (A) NeuN immunostaining of 40-μm brain sections showed a prominent decrease in staining in the hippocampal CA3 region in hAPP/sCrry compared with single transgenic or nontransgenic (Non-tg) mice. The four panels in the bottom right corner show the CA3c subregion of the hAPP (A), sCrry (C), Non-tg (N), and the hAPP/sCrry (A/C) mouse in the upper right corner at higher magnification. (B) The number of NeuN-immunoreactive (IR) neurons in the CA2/CA3 region of the hippocampus was strongly reduced in hAPP/sCrry mice compared with littermate controls. Bars represent mean ± SEM from three sections per mouse and 5–8 mice per genotype. *, P < 0.05 by Tukey–Kramer test. (C) Relative numbers of NeuN-positive neurons in the CA2/CA3 region of the hippocampus of hAPP (○) and hAPP/sCrry (•) mice plotted against the percent area occupied by Aβ immunoreactive deposits (3D6 antibody). (D) Dendritic integrity determined as percent area occupied by MAP-2 immunoreactive dendrites in the stratum radiatum of the CA3 subfield of the hippocampus was reduced in hAPP and hAPP/sCrry mice. Bars represent mean ± SEM from 5–8 mice per genotype. *, P < 0.05; **, P < 0.01 by Tukey–Kramer test. (E) Microglial activation determined as the relative area of F4/80 immunoreactive products in the hippocampus or the midfrontal cortex was reduced in hAPP/sCrry (black bars) compared with hAPP (white bars) mice. Bars are mean ± SEM from 5–8 mice per group. **, P = 0.008 by t test. (F and G) Microglia surrounding amyloid plaques (indicated by asterisks) appear more activated and express more F4/80 immunoreactivity in hAPP (F) than in hAPP/sCrry (G) mice. [Scale bars: 100 μm (A), 20 μm (F and G).]
Fig 5.
Fig 5.
Accumulation of degenerating neurons in hAPP/sCrry mice. Brain sections from 12-month-old nontransgenic (A, C, and D) or hAPP/sCrry mice (B and E–G) were stained with toluidine blue and viewed by light microscopy (A and B) or analyzed by electron microscopy (C–G). (A and B) Dentate gyrus of a nontransgenic (A) or hAPP/sCrry mouse (B) with darkly stained degenerating cells (arrow heads). (C and D) Normal appearing neurons (asterisks) in the dentate gyrus (C) and neuropil (D) in the hippocampal CA3 region of a nontransgenic mouse. (E) Degenerating neurites (arrows) in the CA3 region of the hippocampus in a hAPP/sCrry mouse. (F and G) Neurons with different degrees of electron density (arrow heads) indicating different degrees of degeneration in the dentate gyrus (F) or CA3 pyramidal layer (G). Asterisks mark healthy-appearing neurons. [Scale bars: 25 μm (A and B), 10 μm (C–F), 3 μm (G).]

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