IFN-gamma promotes complement expression and attenuates amyloid plaque deposition in amyloid beta precursor protein transgenic mice - PubMed (original) (raw)

IFN-gamma promotes complement expression and attenuates amyloid plaque deposition in amyloid beta precursor protein transgenic mice

Paramita Chakrabarty et al. J Immunol. 2010.

Abstract

Reactive gliosis surrounding amyloid beta (Abeta) plaques is an early feature of Alzheimer's disease pathogenesis and has been postulated to represent activation of the innate immune system in an apparently ineffective attempt to clear or neutralize Abeta aggregates. To evaluate the role of IFN-gamma-mediated neuroinflammation on the evolution of Abeta pathology in transgenic (Tg) mice, we have expressed murine IFN-gamma (mIFN-gamma) in the brains of Abeta precursor protein (APP) Tg mice using recombinant adeno-associated virus serotype 1. Expression of mIFN-gamma in brains of APP TgCRND8 mice results in robust noncell autonomous activation of microglia and astrocytes, and a concomitant significant suppression of Abeta deposition. In these mice, mIFN-gamma expression upregulated multiple glial activation markers, early components of the complement cascade as well as led to infiltration of Ly-6c positive peripheral monocytes but no significant effects on APP levels, APP processing or steady-state Abeta levels were noticed in vivo. Taken together, these results suggest that mIFN-gamma expression in the brain suppresses Abeta accumulation through synergistic effects of activated glia and components of the innate immune system that enhance Abeta aggregate phagocytosis.

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Figures

Fig 1. rAAV1-mIFNγ expression in TgCRND8 mice following neonatal intracerebroventricular injection results in extensive induction of microgliosis and astrogliosis

A-F. rAAV1-mIFNγ or rAAV1-EGFP (Control mice) was injected into the cerebral ventricles of TgCRND8 mice on neonatal day P2 and sacrificed after 5 months (P2→5mo). Representative images of Iba-1 immunoreactivity in paraffin embedded whole brain sections (A, B) and higher magnifications of the hippocampus (lower panels, C-F) is shown. Abundant activated microglia displaying hypertrophic processes are present in mIFNγ expressing mice (B, D, F) compared to EGFP expressing control mice (A, C, E). Scale Bar, 600µm (A, B), 150µm (C, D) and 25µm (E, F). _n_=10/group. G-L. Representative images of GFAP immunoreactivity in paraffin embedded sections of P2→5 month old TgCRND8 mice expressing mIFNγ or EGFP is depicted. Whole brain sections (G, H) along with higher magnification pictures (lower panels, I-L) showing detailed morphology of the activated astrocytes in and around the corresponding hippocampus are shown. Abundant astrocytes are evident in mIFNγ expressing mice (H, J, L) compared to EGFP expressing control mice (G, I, K). Scale Bar, 600µm (G, H), 150µm (I, J) and 25µm (K, L). _n_=10/group. M. Quantitation of Iba-1 and GFAP immunoreactivity burden (% area) in paraffin embedded sections of P2→5 month old TgCRND8 mice expressing mIFNγ or EGFP as control. (_n_=5/group, *p<0.05). N. Levels of mIFNγ were increased in mIFNγ expressing P2→5 month old TgCRND8 mice brains compared to age-matched controls. mIFNγ protein levels were analyzed using RIPA buffer solubilized brain lysates by sandwich ELISA. (_n_=5/group, *p<0.05). O-P. Representative immunoblot showing increased levels of CD11b and GFAP levels in P2→5 month old mIFNγ expressing TgCRND8 mice compared to controls (O). β-Actin has been used as a loading control. Intensity analysis of CD11b and GFAP immunoreactive bands normalized to β actin is depicted (P). (_n_=3/group, *p<0.05).

Fig 2. Significant attenuation of amyloid deposition in mIFNγ expressing (P2→5mo) TgCRND8 mice

A-H. rAAV1-mIFNγ or rAAV1-EGFP was injected into the cerebral ventricles of TgCRND8 mice on neonatal day P2 and sacrificed after 5 months (P2→5mo). mIFNγ expressing TgCRND8 mice (A, C-E) were analyzed along with age-matched EGFP expressing mice (B, F-H, Control). Representative sections of the whole brain (A-B) as well as hippocampus (C-H) from 3 mice from each group is shown following pan Aβ immunostaining. Scale Bar, 600µm (A-B), 150µm (C-H). _n_=10–12/group). I-J. Image analysis of amyloid plaque immunoreactivity shows a significant decrease in Aβ plaque burdens in the forebrain (I) and hippocampus (J) of mIFNγ expressing mice compared to EGFP expressing control mice (_n_=10–12/group, *p<0.05). K-L. Biochemical analyses of Aβ42 and Aβ40 levels in P2→5 month old mIFNγ expressing TgCRND8 mice compared to EGFP expressing age matched controls. Both SDS-soluble and SDS-insoluble (FA fraction) Aβ42 and Aβ40 levels in the forebrain of mIFNγ injected mice were significantly reduced compared to control mice (_n_=10/group, *p<0.05 and **p<0.05).

Fig 3. Amyloid deposition is suppressed following acute focal expression of mIFNγ in the hippocampus of TgCRND8 mice

A-I. 4 month old TgCRND8 mice were stereotaxically injected in the hippocampus with either rAAV1-mIFNγ or rAAV1-EGFP and sacrificed after 6 weeks. Representative brain sections stained with pan Aβ antibody depict attenuation of Aβ deposition in mIFNγ expressing mice (D-F) compared to EGFP injected controls (A-C) in the immediate vicinity of the injection site. Unmanipulated 4 month old TgCRND8 brains, dissected at the same level, are depicted (G-I). Scale Bar, 150µm. _n_=5/group. J. Aβ plaque burden analysis shows a significant decrease in amyloid deposition in 5.5 month old mIFNγ expressing mice compared to EGFP expressing age-matched control mice but no change compared to unmanipulated 4 month old TgCRND8. (_n_=5/group, *p<0.05). K-L. Biochemical analyses of Aβ42 and Aβ40 levels by ELISA show significant reductions in both SDS-soluble (H) and SDS-insoluble FA fractions (I) in mIFNγ expressing mice compared to controls (_n_=5/group, *p<0.05 and **p<0.05).

Fig 4. APP processing is not significantly altered in mIFNγ expressing P2→5 month old TgCRND8 mice

A-B. Representative anti CT20 immunoblot depicting APP levels in mIFNγ expressing P2→5 month old TgCRND8 and age-matched control mice (A). Intensity analysis of anti CT20 immunoreactive APP bands normalized to β-actin reveal no significant changes in APP levels in mIFNγ expressing TgCRND8 mice compared to age-matched controls (B). _n_=4/group. C-D. Representative immunoblot showing CTFα (anti-CT20) and CTFβ (anti-82E1) levels in P2→5 month old TgCRND8 mice expressing mIFNγ or EGFP (C). Intensity analysis of CTFα and CTFβ bands normalized to β-actin reveal no significant changes in P2→5 month old TgCRND8 mice expressing mIFNγ compared to age-matched controls (D). _n_=3/group. E-F. Representative anti CT20 immunoblot showing APP or CTFα levels in mIFNγ expressing 4→5.5 month old TgCRND8 mice and age-matched controls (E). Intensity analysis of APP and CTFα levels normalized to β-actin show no significant changes in 4→5.5 month old TgCRND8 mice expressing mIFNγ compared to age-matched controls (F). _n_=5/group.

Fig 5. mIFNγ expression leads to alterations in glial activation markers and pro-inflammatory cytokines

Expression of glial activation markers (A) and cytokines (B) were determined in P2→5 month old mIFNγ expressing TgCRND8 mice compared to EGFP expressing age-matched transgenic controls using real time Q-PCR. Relative quantitation of mRNA transcript levels was performed using the comparative cycle threshold method. The expression levels of different genes were normalized using β-actin levels from the corresponding samples. Data, expressed as relative units of mRNA expression, represents averaged fold change values obtained from mIFNγ expressing mice, relative to averaged values obtained from EGFP expressing mice. The horizontal line represents the reference point used for relative mRNA analysis. Error bars indicate SEM. (_n_=4/group, *p<0.05).

Fig 6. mIFNγ expression leads to increased levels of complement protein C3 in 5 month old TgCRND8 mice

A. Increased mRNA transcript levels of complement protein C1q, C3, and C4a as determined by Q-PCR in P2→5 month old TgCRND8 mice expressing mIFNγ. Relative quantitation of mRNA expression was performed using the comparative cycle threshold method. The expression levels of different genes were normalized using β-actin levels from the corresponding samples. Data, expressed as relative units of mRNA expression, represents averaged fold change values obtained from mIFNγ expressing mice, relative to averaged values obtained from EGFP expressing mice. The horizontal line represents the reference point used for relative mRNA analysis. Error bars indicate SEM. (_n_=4/group, *p<0.05). B-C. Representative immunoblot depicting complement C3 protein levels in P2→5 month old TgCRND8 mice expressing mIFNγ or EGFP (B). Quantitative analysis of anti C3 positive immunoreactive band normalized to β-actin shows significantly increased levels of C3 in P2→5 month TgCRND8 mice expressing mIFNγ compared to age-matched controls (C). (_n_=4/group, *p<0.05). D-I. Representative images depicting anti-C3 immunofluorescence staining (red stain) colocalizing with β-tubulin immunoreactive neurons (green stain) in the hippocampus of mIFNγ expressing P2→5 month old TgCRND8 mice compared to controls. Blue represents the DAPI stained nuclei. (magnification, 600×). _n_=3/group.

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IFN-gamma promotes complement expression and attenuates amyloid plaque deposition in amyloid beta precursor protein transgenic mice - PubMed (original) (raw)