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Enhanced antigen retrieval of amyloid ? immunohistochemistry: re-evaluation of amyloid ? pathology in Alzheimer disease and its mouse model.

ABSTRACT: Senile plaques, extracellular deposits of amyloid ? peptide (A?), are one of the pathological hallmarks of Alzheimer disease (AD). As the standard immunohistochemical detection method for A? deposits, anti-A? immunohistochemistry combined with antigen retrieval (AR) by formic acid (FA) has been generally used. Here, we present a more efficient AR for A? antigen. On brain sections of AD and its mouse model, a double combination of either autoclave heating in EDTA buffer or digestion with proteinase K plus FA treatment reinforced A? immunoreactivity. A further triple combination of digestion with proteinase K (P), autoclave heating in EDTA buffer (A), and FA treatment (F), when employed in this order, gave a more enhanced immunoreactivity. Our PAF method prominently visualized various forms of A? deposits in AD that have not been clearly detected previously and revealed numerous minute-sized plaques both in AD and the mouse model. Quantification of A? loads showed that the AR effect by the PAF method was 1.86-fold (in the aged human brain) and 4.64-fold (in the mouse brain) higher than that by the FA method. Thus, the PAF method could have the potential to be the most sensitive tool so far to study A? pathology in AD and its mouse model.

SUBMITTER: Kai H

PROVIDER: S-EPMC3524564 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Enhanced antigen retrieval of amyloid β immunohistochemistry: re-evaluation of amyloid β pathology in Alzheimer disease and its mouse model.

Kai Hideaki H Shin Ryong-Woon RW Ogino Koichi K Hatsuta Hiroyuki H Murayama Shigeo S Kitamoto Tetsuyuki T

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 20120721 10

Senile plaques, extracellular deposits of amyloid β peptide (Aβ), are one of the pathological hallmarks of Alzheimer disease (AD). As the standard immunohistochemical detection method for Aβ deposits, anti-Aβ immunohistochemistry combined with antigen retrieval (AR) by formic acid (FA) has been generally used. Here, we present a more efficient AR for Aβ antigen. On brain sections of AD and its mouse model, a double combination of either autoclave heating in EDTA buffer or digestion with proteina ...[more]

PMID: 22821668

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Project description:IntroductionAlzheimer pathology (AD) is characterized by the deposition of amyloid beta (Aβ) and chronic neuroinflammation, with the NLRP3 inflammasome playing a significant role. This study demonstrated that the OCD drug fluvoxamine maleate (FXN) can potently ameliorate AD pathology in 5XFAD mice by promoting autophagy-mediated clearance of Aβ and inhibiting the NLRP3 inflammasome.MethodsWe used mice primary astrocytes to establish the mechanism of action of FXN against NLRP3 inflammasome by using various techniques like ELISA, Western blotting, confocal microscopy, Immunofluorescence, etc. The anti-AD activity of FXN was validated in transgenic 5XFAD mice following two months of treatment. This was followed by behavior analysis, examination of inflammatory and autophagy proteins and immunohistochemistry analysis for Aβ load in the hippocampi.ResultsOur data showed that FXN, at a low concentration of 78 nM, induces autophagy to inhibit NF-κB and the NLRP3 inflammasome, apart from directly inhibiting NLRP3 inflammasome in primary astrocytes. FXN activated the PRKAA2 pathway through CAMKK2 signaling, leading to autophagy induction. It inhibited the ATP-mediated NLRP3 inflammasome activation by promoting the autophagic degradation of NF-κB, resulting in the downregulation of pro-IL-1β and NLRP3. The anti-NLRP3 inflammasome effect of FXN was reversed when autophagy was inhibited by either genetic knockdown of the PRKAA2 pathway or pharmacological inhibition with bafilomycin A1. Furthermore, FXN treatment led to improved AD pathology in 5XFAD mice, resulting in significant improvements in various behavioral parameters such as working memory and neuromuscular coordination, making their behavior more similar to that of wild-type animals. FXN improved behavior in 5XFAD mice by clearing the Aβ deposits from the hippocampi and significantly reducing multiple inflammatory proteins, including NF-κB, GFAP, IBA1, IL-1β, TNF-α, and IL-6, which are associated with NF-κB and NLRP3 inflammasome in the brain. Moreover, these changes were accompanied by increased expression of autophagic proteins.DiscussionOur data suggest that FXN ameliorates AD pathology, by simultaneously targeting two key pathological features: Aβ deposits and neuroinflammation. As an already approved drug, FXN holds potential as a candidate for human studies against AD.

Dataset Information

Enhanced antigen retrieval of amyloid ? immunohistochemistry: re-evaluation of amyloid ? pathology in Alzheimer disease and its mouse model.

Publications

Enhanced antigen retrieval of amyloid β immunohistochemistry: re-evaluation of amyloid β pathology in Alzheimer disease and its mouse model.

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