Recombinant Integrin ß1 Signal Peptide Blocks Gliosis Induced by Aß Oligomers.

Glial cells participate actively in the early cognitive decline in Alzheimer's disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aß) oligomers and integrin ß1. Here, we have generated four recombinant peptides including the extracellular domain of integrin ß1, and evaluated their capacity both to bind in vitro to Aß oligomers and to prevent in vivo Aß oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin ß1 that binds to Aß oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin ß1 N-terminal domain. This recombinant integrin ß1 signal peptide prevented Aß oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin ß1 signal peptide combined with or without Aß oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin ß1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aß oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aß oligomer/integrin ß1 signaling.

Amyloid ß-induced astrogliosis is mediated by ß1-integrin via NADPH oxidase 2 in Alzheimer's disease.

Astrogliosis is a hallmark of Alzheimer'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-ß modulates ß1-integrin activity and triggers NADPH oxidase (NOX)-dependent astrogliosis in vitro and in vivo. Amyloid-ß oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by ß1-integrin in cultured astrocytes. This mechanism promotes ß1-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 ß1-integrin in reactive astrocytes which correlates with the amyloid ß-oligomer 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 ß1-integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and ß1-integrin were significantly associated with increased amyloid-ß load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid ß oligomers with ß1-integrin which in turn leads to enhancing ß1-integrin and NOX2 activity via NOX-dependent mechanisms. These observations may be relevant to AD pathophysiology.

A trifluoromethyl analogue of celecoxib exerts beneficial effects in neuroinflammation.

Celecoxib is a selective cyclooxygenase-2 (COX2) inhibitor. We have previously shown that celecoxib inhibits experimental autoimmune encephalomyelitis (EAE) in COX-2-deficient mice, suggestive for a mode of action involving COX2-independent pathways. In the present study, we tested the effect of a trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2 inhibitory activity in two models of neuroinflammation, i.e. cerebellar organotypic cultures challenged with LPS and the EAE mouse model for multiple sclerosis. TFM-C inhibited secretion of IL-1ß, IL-12 and IL-17, enhanced that of TNF-α and RANTES, reduced neuronal axonal damage and protected from oxidative stress in the organotypic model. TFM-C blocked TNF-α release in microglial cells through a process involving intracellular retention, but induced TNF-α secretion in primary astrocyte cultures. Finally, we demonstrate that TFM-C and celecoxib ameliorated EAE with equal potency. This coincided with reduced secretion of IL-17 and IFN-γ by MOG-reactive T-cells and of IL-23 and inflammatory cytokines by bone marrow-derived dendritic cells. Our study reveals that non-coxib analogues of celecoxib may have translational value in the treatment of neuro-inflammatory conditions.

Ca(2+) -dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid ß-treated astrocytes and in a model of Alzheimer's disease.

Neurotoxic effects of amyloid ß peptides are mediated through deregulation of intracellular Ca(2+) homeostasis and signaling, but relatively little is known about amyloid ß modulation of Ca(2+) homeostasis and its pathological influence on glia. Here, we found that amyloid ß oligomers caused a cytoplasmic Ca(2+) increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca(2+) release. Furthermore, amyloid ß peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2α phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP3 receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid ß 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 ß 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 ß oligomers disrupt ER Ca(2+) homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.