Alzheimer’s disease (AD) brains are characterized by fibrillar amyloid-β (Aβ) peptide containing plaques and associated reactive microglia. The proinflammatory phenotype of the microglia suggests that they may negatively affect disease course and contribute to behavioral decline. This hypothesis predicts that attenuating microglial activation may provide benefit against disease. Prior work from our laboratory and others has characterized a role for the transcription factor, nuclear factor of activated T cells (NFAT), in regulating microglial phenotype in response to different stimuli, including Aβ peptide. We observed that the NFATc2 isoform was the most highly expressed in murine microglia cultures, and inhibition or deletion of NFATc2 was sufficient to attenuate the ability of the microglia to secrete cytokines. In order to determine whether the NFATc2 isoform, in particular, was a valid immunomodulatory target in vivo, we crossed an NFATc2−/− line to a well-known AD mouse model, an AβPP/PS1 mouse line. As expected, the AβPP/PS1 × NFATc2−/− mice had attenuated cytokine levels compared to AβPP/PS1 mice as well as reduced microgliosis and astrogliosis with no effect on plaque load. Although some species differences in relative isoform expression may exist between murine and human microglia, it appears that microglial NFAT activity is a viable target for modulating the proinflammatory changes that occur during AD.
Our data suggests that the antibody therapy was able to reduce microgliosis, astrogliosis, and synaptic changes in the APP/PS1 mice compared to isotype control injections without changing amyloid-β plaque load. Interestingly, both isotype control and antibody therapy also reduced the number of proinflammatory cytokines in the spleen although changes in the brain were less robust. The anti-CD49d and isotype control treatments also reduced CD4 immunoreactivity in the brains, suggesting a possible mechanism for attenuation of inflammation in the brain. This data suggests that it is indeed feasible to alter the immune component of AD brain changes using a clinically feasible strategy of delivering a particular subtype of IgG or epitope selective antibodies that target infiltration of the peripheral immune system.
Antigen retrieval is a standard procedure to enhance immunohistochemical detection. However, among the many choices of techniques available for antigen retrieval, it is important to choose a method that works specifically for the antibody of interest. The small calcium binding protein, Iba1, has been well characterized as a microglia specific marker useful for identifying both resting and activated populations (ito etal., 1998[1]). In this study, we tested whether antigen retrieval methods would increase the sensitivity or improve the morphologic visualization of Iba1 immunoreactive microglia in the brains of wild type C57BL/6 mice and an APP/PS1 mouse model of Alzheimer's disease (AD). A more sensitive detection method might allow for better quantitation of microglial changes during disease. We modified a protocol which used three different methods and their combination for retrieving specifically anti-Ab immunoreactivity in AD mouse brains to determine whether it improved Iba1 staining (Kai et al., 2012; Murayama et al., 1999). The following modifications were made to the original protocol:
We boiled the free floating brain sections or slide mounted brain sections in 10 mM EDTA solution (pH 6.0) in a secondary water bath instead of autoclaving for attempting Iba1 antigen retrieval.We used a 15 min, 0.25% trypsin-EDTA treatment instead of protease K for attempting Iba1 antigen retrieval.We immunostained with anti-Iba1 antibody as our primary interest but also stained some sections in parallel with 4G8 antibody for anti-Aβ staining comparison.
Iba1 immunoreactivity was best enhanced by boiling in the low pH EDTA solution for both free floating and slide mounted tissues.
We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eight rrn operons (rrna-rrnh) of Vibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O 139 outbreak. ISR classes 'a' and 'g' were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O 139 serogroup and post-O 139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.
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