Microglial C5aR (CD88) expression correlates with amyloid-beta deposition in murine models of Alzheimer's disease

Abstract

Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by the accumulation of amyloid-beta protein and neuronal loss, is the leading cause of age-related dementia in the world today. The disease is also associated with neuroinflammation, robust activation of astrocytes and microglia, and evidence of activation of the complement system, localized with both fibrillar amyloid-beta (fAbeta) plaques and tangles. The observations are consistent with a complement-dependent component of AD progression. We have previously shown that inhibition of the major complement receptor for C5a (CD88) with the antagonist PMX205 results in a significant reduction in pathology in two mouse models of AD. To further characterize the role of complement in AD-related neuroinflammation, we examined the age- and disease-associated expression of CD88 in brain of transgenic mouse models of AD and the influence of PMX205 on the presence of various complement activation products using flow cytometry, western blot, and immunohistochemistry. CD88 was found to be up-regulated in microglia, in the immediate vicinity of amyloid plaques. While thioflavine plaque load and glial recruitment is significantly reduced after treatment with PMX205, C1q remains co-localized with fAbeta plaques and C3 is still expressed by the recruited astrocytes. Thus, with PMX205, potentially beneficial activities of these early complement components may remain intact, while detrimental activities resulting from C5a-CD88 interaction are inhibited. This further supports the targeted inhibition of specific complement mediated activities as an approach for AD therapy.

Figure 1. Monoclonal rat anti-mouse CD88 antibody clone 10/92 specifically recognizes CD88 in purified microglia cultures

A) Microglia from wild type and CD88-/- mice were stained with clone 10/92 monoclonal rat anti-mouse CD88 antibody and analyzed by flow cytometry. Grey shading represents isotype rat IgG control antibody. (B) Wild type or CD88-/- microglial and neuronal cell lysates (10μg per lane, under reduced conditions) were subjected to SDS PAGE followed by transfer to PDVF membrane, and subsequently probed with the rat anti-mouse CD88 (clone 10/92) antibody. Representative images of primary microglial cultures from CD88-/- (D) and CD88+/+ (F) mice stained with clone 10/92 monoclonal anti-mouse CD88. (C, E are corresponding phase images). Scale bar: 50 μm. (G) RNA extracted from wild type and CD88-/- microglia and mature neurons (14 days), was analyzed using RT-PCR.

Figure 2. Anti-CD88 antibodies show prominent reactivity in plaque-surrounding cells with some cross reactivity in non-transgenic CD88-/- mouse brain tissue

Brain tissues from Balb/c (A), CD88-/- (B) and Tg2576 AD mouse model (C) were stained using monoclonal rat anti-mouse CD88 clone 10/92 (Serotec). CD88 reactivity was observed in the vicinity of Aβ plaques in Tg2576 mice (C). No reactivity was observed in the absence of the primary monoclonal rat anti-mouse CD88 (Tg2576, D). Scale bar: 50μm. (E) Brain extracts from C57/B6 wild type (WT), CD88-/- or Tg2576 model mouse brains (40μg per lane, under reduced conditions) all contain a detectable band at 45kDa when probed with the anti-mouse CD88 clone 10/92 antibody. (F) Immunoprecipitation of wild type and CD88-/- brain extracts with BD Pharmingen C1150-32 anti-CD88 antibody or control rabbit IgG, followed by Western blot analysis probed with anti-CD88 clone 10/92 demonstrates shared reactivity of the anti-CD88 antibodies with a 45kDa protein not found in CD88-/- extracts. (H,I) CD88 immunostaining detected surrounding plaques of Arc48 mice (H) is not present in the Arc48 CD88-/- (I).

Figure 3. CD88 expression correlates with increased pathology in AD mouse models

CD88 (red, clone 10/92) is associated with fibrillar amyloid-β plaque (thioflavine, green) in the cortex of (A) Tg2576 mice at 3,10, and 15 months of age and (B) Arc48 mice at 2, 6 and 13 months. Scale bar: 50μm.

Figure 4. CD88 co-localizes with microglia but not with astrocytes in AD mouse models

(A) Reactive astrocytes, stained with GFAP (green), found in the immediate vicinity of fibrillar amyloid-β plaques do not co-localize with CD88 (red) reactivity in Arc48 mice at 9m of age. (B) CD88 (red) co-localizes with Iba-1 (green) positive microglia surrounding plaques in the Tg2576 model at 15m of age. (C) CD88 (red) co-localizes with CD45 (green) positive microglia surrounding plaques in Arc48 mice at 6m of age. Scale bar: 50 μm.

Figure 5. CD88 can be polarized in membrane processes of microglia, but not astrocytes, adjacent to plaques

Confocal image of CD88 (red) in the Arc48 mouse at 6m of age shows predominant CD88 localization in microglia processes adjacent to plaques in microglia (Iba-1, green) (A), but is not detected in astrocytes (GFAP, green) (B) surrounding amyloid deposits. Scale bar: 10μm.

Figure 6. Treatment with PMX 205 does not alter CD88 expression in plaque associated microglia

Tg2576 mice (15 mo), both untreated (A) and treated with 20 μg/ml PMX205 in the drinking water (B) for 12 weeks were perfused and coronal sections were stained with anti-CD88 antibody (clone 10/92) (red) as described in Materials and Methods. Scale bar: 50 μm.

Figure 7. PMX205 does not inhibit C1q deposition on plaques

Representative images of co-localization of C1q (brown) (A,D) and thioflavine (green) (B,E) in untreated (left) and PMX205 treated (right) Tg2576 mice at 15m of age. Bottom panels (C,F) represent respective merged images. Scale bar: 50 μm.

Figure 8. C3 expression in astrocytes is correlated with plaque pathology in both PMX205 treated and untreated mice

Immunofluorescent co-localization of C3 (red) and GFAP (green) in untreated (A-C) and PMX205 treated (D-F) Tg2576 mice. (A,D: C3; B,E: GFAP; C,F: merge). Scale bar: 50 μm.