Implication of the small GTPase Rac1 in the generation of reactive oxygen species in response to β-amyloid in C6 astroglioma cells

Exogenous application of β-amyloid (Aβ_25-35, a fragment of Aβ_1-42) significantly elevated levels of reactive oxygen species (ROS) in C6 astroglioma cells, as measured by confocal microscopic analysis of H₂O₂-sensitive 2′,7′-dichlorofluorescin fluorescence. Subsequent characterization of the signalling path-way revealed that expression of RacN17, a dominant-negative Rac1 mutant, completely blocked Aβ_25-35-induced generation of ROS, which is indicative of the crucial role played by Rac GTPase in this process. To better understand the downstream mediators affected by Rac, we assessed the degree to which inhibition of cytosolic phospholipase A₂ (cPLA₂) and 5-lipoxygenase (5-LO) contributed to the response and found that inhibition of either enzyme completely blocked Aβ25-35-induced ROS generation, indicating its dependence on arachidonic acid synthesis and metabolism to leukotrienes (e.g. leukotriene B4). Consistent with those findings, Aβ_25-35 Rac-dependently stimulated translocation of 5-LO to the nuclear envelope and increased intracellular levels of leukotriene B₄, while exogenous application of leukotriene B₄ increased intracellular H₂O₂ via BLT, its cell-surface receptor. In addition to the aforementioned downstream mediators, inhibition of phosphoinositide 3-kinase (PI 3-kinase), an enzyme situated upstream of Rac, also completely blocked Aβ_25-35-induced H₂O₂ generation. Our findings thus demonstrate that PI 3-kinase, Rac, cPLA₂ and 5-LO are all essential components of the β-amyloid signaling cascade leading to generation of ROS.