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Daniel W. Lapp, Samuel S. Zhang, Colin J. Barnstable; Reduction Of Mitochondrial ROS Production By UCP2 Is A Potential Mechanism For The Neuroprotective Effects Of Cytokine-induced Stat3 Activation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1861.
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© ARVO (1962-2015); The Authors (2016-present)
Preventing reactive oxygen species (ROS) production by mitochondria is very important in protecting neural cell death in the retina. The level of ROS production depends on the mitochondrial membrane potential, which in turn is regulated by the activity of UCP2, a mitochondrial uncoupling protein. Activation of Stat3 is critical for the observed neuroprotective effects of the cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). Here we elucidate the effect of STAT3 activation by CNTF and LIF on mitochondrial membrane potential, ROS production and UCP2.
Effects of CNTF or LIF were evaluated using glial cell cultures isolated from P4-P7 mice and grown in DMEM/F12 with serum. Animal protocols were in accordance with ARVO / IACUC guidelines. Purity of cultures was evaluated by immunocytochemistry. Stat3 was activated using 20 ng/mL LIF or 25 ng/mL CNTF. Stat3 phosphorylation was inhibited with 100 µM of the JAK2 inhibitor AG490. Phosphorylation of Stat3 by CNTF was assessed using western blotting. The effect of CNTF or LIF treatment on ROS was evaluated using the dye CMH2X. The effect of 24-48 hours of CNTF or LIF treatment on mitochondrial membrane potential was evaluated using the dual color dye JC-1. Stat3 was activated with CNTF or inhibited with AG490. Resulting changes in UCP2 mRNA were measured using quantitative real-time PCR.
Flow cytometry experiments using CMH¬2X demonstrate that 24 hr of treatment with CNTF or LIF reduced ROS in glial cells. Treatment with LIF or CNTF for 24 hours reduced mitochondrial membrane potential as determined by JC-1. An increase in UCP2 mRNA of approximately 50% is seen upon 24 hr treatment of primary glial cultures with CNTF. Treatment of glial cultures with AG490 for 24 hours decreased UCP2 transcripts by two-thirds.
CNTF and LIF cause a decrease in endogenous ROS in glia in vitro, and a concurrent decrease in mitochondrial membrane potential. A CNTF-induced increase in the mitochondrial uncoupling protein UCP2 may be responsible for the observed alterations in mitochondrial membrane potential and suggests a potential downstream mechanism for neuroprotection by Stat3 activation.
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