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Mitochondrial dysfunction is implicated in age-related degenerative disorders such as Alzheimer's disease (AD). Maintenance of mitochondrial dynamics is essential for regulating mitochondrial function. Ab oligomers (AbOs), the typical cause of AD, lead to mitochondrial dysfunction and neuronal loss. AbOs have been shown to induce mitochondrial fragmentation, and their inhibition suppresses mitochondrial dysfunction and neuronal cell death. Oxidative stress is one of the earliest hallmarks of AD. Cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by disrupting the antioxidant system, including Prx2. Cdk5 is also regarded as a modulator of mitochondrial fission; however, a precise mechanistic link between Cdk5 and mitochondrial dynamics is lacking. We estimated mitochondrial morphology and alterations in mitochondrial morphologyrelated proteins in Neuro-2a (N2a) cells stably expressing the Swedish mutation of amyloid precursor protein (APP), which is known to increase AbO production. We demonstrated that mitochondrial fragmentation by AbOs accompanies reduced mitofusin 1 and 2 (Mfn1/2) levels. Interestingly, the Cdk5 pathway, including phosphorylation of the Prx2-related oxidative stress, has been shown to regulate Mfn1 and Mfn2 levels. Furthermore, Mfn2, but not Mfn1, over-expression significantly inhibits the AbO-mediated cell death pathway. Therefore, these results indicate that AbO-mediated oxidative stress triggers mitochondrial fragmentation via decreased Mfn2 expression by activating Cdk5-induced Prx2 phosphorylation.

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and dyyu10@kribb.re.kr 1 These authors contributed equally to this study.Abbreviations used: ARE, antioxidant response element; CM-H 2 DCFDA, 5,6-chloromethyl-2¢,7¢-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; IL, interleukin; iNOS, inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; JNK1-DN, JNK1 dominant-negative; LPS, lipopolysaccharide; NAC, N-acetyl-L-cysteine; RNS, reactive nitrogen species; NO, nitric oxide; Nox, NADPH oxidase; PBS, phosphate buffered saline; PBST, PBS containing 0.02% Tween-20; Prx, peroxiredoxin; ROS, reactive oxygen species; semi-qPCR, Semi-quantitative PCR; SMT, S-methylisothiourea sulfate; SNP, sodium nitroprusside; TBS, Tris-buffered saline; TNF, tumor necrosis factor. AbstractReactive oxygen species (ROS) actively participate in microglia-mediated pathogenesis as pro-inflammatory molecules. However, little is known about the involvement of specific antioxidants in maintaining the microglial oxidative balance. We demonstrate that microglial peroxiredoxin (Prx) 5 expression is up-regulated by lipopolysaccharide (LPS) through activation of the ROS-sensitive signaling pathway and is involved in attenuation of both microglial activation and nitric oxide (NO) generation. Unlike in stimulation of oxidative insults with paraquat and hydrogen peroxide, Prx V expression is highly sensitive to LPS-stimulation in microglia. Reduction of ROS level by treatment with either NADPH oxidase inhibitor or antioxidant ablates LPS-mediated Prx V up-regulation in BV-2 microglial cells and is closely associated with the activation of the c-jun Nterminal kinase (JNK) signaling pathway. This suggests the involvement of ROS/JNK signaling in LPS-mediated Prx V induction. Furthermore, NO induces Prx V up-regulation that is ablated by the addition of inducible nitric oxide synthase inhibitor or deleted mutation of inducible nitric oxide synthase in LPS-stimulated microglia. Therefore, these results suggest that Prx V is induced by cooperative action among the ROS, RNS, and JNK signaling cascades. Interestingly, knockdown of Prx V expression causes the acceleration of microglia activation, including augmented ROS generation and JNKdependent NO production. In summary, we demonstrate that Prx V plays a key role in the microglial activation process through modulation of the balance between ROS/NO generation and the corresponding JNK cascade activation.