SummaryNeurotoxic effects of amyloid b peptides are mediated through deregulation of intracellular Ca 2+ homeostasis and signaling, but relatively little is known about amyloid b modulation of Ca 2+ homeostasis and its pathological influence on glia. Here, we found that amyloid b oligomers caused a cytoplasmic Ca 2+ increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca 2+ release. Furthermore, amyloid b peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2a phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP 3 receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid b oligomers triggered overexpression of GFAP and GRP78 in astrocytes of the hippocampal dentate gyrus. These data were validated in a triple-transgenic mouse model of Alzheimer's disease (AD). Overexpression of GFAP and GRP78 in the hippocampal astrocytes correlated with the amyloid b oligomer load in 12-month-old mice, suggesting that this parameter drives astrocytic ER stress and astrogliosis in vivo. Together, these results provide evidence that amyloid b oligomers disrupt ER Ca 2+ homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.

SummaryAstrogliosis is a hallmark of Alzheimer 0 s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid-b modulates b1-integrin activity and triggers NADPH oxidase (NOX)-dependent astrogliosis in vitro and in vivo. Amyloid-b oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by b1-integrin in cultured astrocytes. This mechanism promotes b1-integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple-transgenic AD mouse model shows increased levels of GFAP, NOX2, and b1-integrin in reactive astrocytes which correlates with the amyloid boligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and b1-integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and b1-integrin were significantly associated with increased amyloid-b load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid b oligomers with b1-integrin which in turn leads to enhancing b1-integrin and NOX2 activity via NOX-dependent mechanisms. These observations may be relevant to AD pathophysiology.