These results suggested that iNOS signaling might make a small contribution to cytokine stimulated increases in astrocytic secreted selleck chemicals Ruxolitinib Ab, but it may do so via a mechanism that is independent of effects on APP and BACE1 expression. Ab42 increases astrocytic BACE1, APP, and b secretase processing It has been posited that AD may involve a vicious cycle that becomes self perpetuating once it is started. However, direct evidence for this hypothesis has been Inhibitors,Modulators,Libraries difficult to obtain. Given that we observed that Ab secretion was increased in cytokine stimulated astro cytes, and that astrocytic cytokine release was induced by Ab, we investigated the possibility of an astrocytic vicious Inhibitors,Modulators,Libraries cycle involving an Ab stimulated feed forward loop.
Specifically, we sought to determine whether oligomers and fibrils of Ab42, the putative pathogenic agent in AD, could elevate endogenous APP, BACE1, and b secretase cleavage of APP in astrocytes. If so, astrocytes might represent a significant source of Ab production Inhibitors,Modulators,Libraries in AD, Inhibitors,Modulators,Libraries and understanding the associated mechanism could potentially identify novel astrocyte specific Ab lowering therapeutic strategies. To gain insight into these questions, we cultured pri mary astrocytes from the brains of neonatal C57BL 6J or Tg2576 mouse pups and then treated astrocyte cul tures with either oligomeric or fibrillar Ab42 prepared as previously described. Following treatment, cell lysates were harvested and analyzed for levels of endo genous APP and BACE1 protein and mRNA, and APPsb, the BACE1 cleaved APP ectodomain fragment.
For C57BL 6J wild type primary astrocytes, APP immunoblots revealed that Inhibitors,Modulators,Libraries both Ab42 oligomers and fibrils stimulated a dramatic 400 500% rise in endogen ous APP protein level after 24 h of Ab42 treatment, as compared to oligomeric or fibrillar vehicle controls. This Ab42 stimulated APP increase remained elevated at 48 h of Ab42 treatment, but APP levels returned to vehicle control levels by 96 h of treat ment. Immunofluorescence microscopy with anti APP antibody 22C11 confirmed this robust increase in astrocytic APP level following 24 h of oligo meric Ab42 treatment. These results sug gested that Ab42, irrespective of its aggregation state, was capable of strongly inducing the expression of endo genous astrocytic APP, at least up to 48 h of exposure under the culture conditions that we tested.
To determine whether the Ab42 stimulated astrocytic APP elevation was potentially the result Carfilzomib mechanism of a transcrip tional mechanism, we grew C57BL 6J primary astrocyte cultures, treated them with Ab42 and then isolated mRNA and measured APP mRNA levels with TaqMan quantitative RT PCR. Since both oligomeric and fibrillar Ab42 caused similar increases of APP level in astrocytes, we focused on Ab42 oligomer treated astrocytes because the mechanisms of APP elevation for both forms of Ab42 seemed likely to be the same.