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, 106 (5), 2080-92

Complement C3 and C4 Expression in C1q Sufficient and Deficient Mouse Models of Alzheimer's Disease

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Complement C3 and C4 Expression in C1q Sufficient and Deficient Mouse Models of Alzheimer's Disease

Jun Zhou et al. J Neurochem.

Abstract

Alzheimer's disease (AD) is a neurodegenerative disease resulting in progressive cognitive decline. Amyloid plaque deposits consisting specifically of beta-amyloid peptides that have formed fibrils displaying beta-pleated sheet conformation are associated with activated microglia and astrocytes, are colocalized with C1q and other complement activation products, and appear at the time of cognitive decline in AD. Amyloid precursor protein (APP) transgenic mouse models of AD that lack the ability to activate the classical complement pathway display less neuropathology than do the APPQ+/+ mice, consistent with the hypothesis that complement activation and the resultant inflammation may play a role in the pathogenesis of AD. Further investigation of the presence of complement proteins C3 and C4 in the brain of these mice demonstrate that both C3 and C4 deposition increase with age in APPQ+/+ transgenic mice, as expected with the age-dependent increase in fibrillar beta-amyloid deposition. In addition, while C4 is predominantly localized on the plaques and/or associated with oligodendrocytes in APPQ+/+ mice, little C4 is detected in APPQ-/- brains consistent with a lack of classical complement pathway activation because of the absence of C1q in these mice. In contrast, plaque and cell associated C3 immunoreactivity is seen in both animal models and, surprisingly, is higher in APPQ-/- than in APPQ+/+ mice, providing evidence for alternative pathway activation. The unexpected increase in C3 levels in the APPQ-/- mice coincident with decreased neuropathology provides support for the hypothesis that complement can mediate protective events as well as detrimental events in this disease. Finally, induced expression of C3 in a subset of astrocytes suggests the existence of differential activation states of these cells.

Figures

Figure 1
Figure 1. C1q deposition is age-dependent in APP Q+/+mice brain, and absence in APPQ−/− mice
A. Quantification of C1q immunostaining in animals at different ages (3mo–16mo). Data points represent group means ± SD (n=4 animals per age group except n=6 for 16 months, 5 fields/animal including cortex and hippocampus). 12m and 16m APP mice C1q staining was significantly different from 9 month old animals (*, p<0.01). B. Western blot analysis of 16mo APPQ+/+ and APPQ−/− brain extracts using a polyclonal anti-mouse C1q antibody as described in Materials and Methods. The C1q immunoreactive band is only seen in APPQ+/+ mice.
Figure 2
Figure 2. APPQ−/− have less C4 reactivity than APPQ+/+ mice
A. Representative pictures of cortex of APPQ+/+ (left) and APPQ−/− (right) mice at different ages showing immunostaining with anti-mouse C4 (C4d) (HBT). Scale bar: 50μm. B. Sections from cortex of 16 mo old double transgenic APPPS1 Q+/+ and APPPS1Q−/− were stained with anti-mouse C4 as in A. C. Quantification of C4 reactivity by image analysis shows that C4d staining in APPQ+/+ (-□-) mice is significantly higher (* p<0.02) than in APPQ−/− (-▲-) at 16 mo. (n=3 or 4 mice for each genotype at each age). There was no reactivity in the wild type B6/SJL (-○-). Data points represent group means ± SD. D. Western blot analysis of brain extracts from 16m B6SJL, APPQ+/+ and APPQ−/− mice probed with polyclonal anti-mouse C4 (Ogata, Table 1) detect C4 activation fragment, C4d at 42,000 Mr.
Figure 3
Figure 3. C4 is not associated with astrocytes or microglia
Immunofluorescent double labeling of C4 (red, Ogata anti mouse C4) and either astrocytes (GFAP, green) (A), or microglia (CD45, green) (B) in cortex of 16m APPQ+/+ mice. Scale bar: 25μm.
Figure 4
Figure 4. C4 colocalizes with oligodendrocytes in APP transgenic mouse brain
Confocal images in brain cortex show colocalization of C4 (Ogata polyclonal anti-mouse C4, red,) with oligodendrocyte cell bodies (anti-CNPase monoclonal, green) in 16mo APPQ+/+ (upper panels). In APPQ−/− (lower panels), C4 immunostaining is lower but also present in oligodendrocyte cell bodies. Right panels are merged confocal images. Scale bar: 25μm.
Figure 5
Figure 5. C3 immunoreactivity is greater in APPQ−/− than in APPQ+/+
Anti-C3 staining (HBT) in cortex of APPQ+/+ (left) and APPQ−/− (right) at 12m and 16m shows increased cellular immunoreactivity in APPQ−/− mice. Scale bar: 50 μm. B. Quantification of C3 immunoreactivity in cortex and hippocampus in APPQ+/+ (-□-), APPQ−/− (-▲-) and B6/SJL (-○-) at 6, 9, 12, 16 mo (6m n=3; 9m n=3; 12m n=4; 16m n=4 each genotype) shows significantly higher levels of C3 (anti-C3, HBT) in APPQ−/− than APP at 16 months (* p<0.0 2). Data points are group means ± SD. C. Anti-C3c (Lambris 2/16) staining in 16m APPQ+/+ (left) and APPQ−/− (right) in plaque-like structures. Scale bar: 50 μm D. Image analysis of anti-C3c (2/16) staining. (n=3 animals each genotype; * p < 0.05). Data shown are group means ± SD. E. Western blot of brain extracts from B6/SJL, APPQ+/+ and APPQ−/− at 16 months probed with anti-mouse C3 antibody (Cappel) shows greater reactivity to anti native C3α chain (115,000 Mr) in the APPQ−/− animals.
Figure 6
Figure 6. C3 colocalizes with astrocytes in APPQ+/+ and APPQ−/−
Confocal images of GFAP (green) (A,D) and C3 (red, HBT) (B,E) double labeling in cortex of 16 mo APPQ+/+ (A,B,C) and APPQ−/− (D,E,F) mice. Note fewer astrocytes surrounding plaques in APPQ−/− mice (D) than in APPQ+/+ (A), but the C3 staining appears stronger (E) than in APPQ+/+ (B) Merge of GFAP and C3 (C,F) demonstrates that astrocytes are positive for C3. Scale bar: 25μm
Figure 7
Figure 7. Activated C3 colocalizes with fibrillar amyloid plaques in both APPQ+/+ and APPQ−/− mice
Representative pictures of 16 mo APPQ+/+ and APPQ−/− sections from cortex that were immunostained with rat anti-mouse C3c (Lambris, 2/11) (red) and with thioflavine (β-sheet structure, green. Scale bar: 25μm.
Figure 8
Figure 8. Schematic diagram of potential therapeutic drug targeting
Fibrillar Aβ activates the classical (and alternative) pathway of complement, resulting in C3b/iC3b deposition on plaques, glial recruitment and generation of neurotoxic molecules, and potential lysis by the Membrane attack complex (MAC/C5b-9) of complement. The red and green arrows signify pathways that are probably detrimental and protective respectively. The yellow arrows represent events that are suggested to occur in response to complement activation in vivo but have yet to be definitively proven. The black dotted lines indicate potential targets for intervention/inhibition of detrimental pathways. Modified from (Tenner 2001).

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