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J. M. Silva, A. Wong, G. A. Cortopassi; The Effect of Mitochondrial Inhibition in Oligodendroglial Cells: A Model for Leber's Hereditary Optic Neuropathy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):940.
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© ARVO (1962-2015); The Authors (2016-present)
Leber's Hereditary Optic Neuropathy (LHON) is caused by point mutations within Complex I (NADH dehydrogenase) of the mitochondrial genome that leads to the neurodegeneration of the retinal ganglion cells and optic nerve as well as axonal demyelination, ultimately resulting in blindness. In a previous microarray study, we demonstrated that mitochondrial diseases can inhibit myelin-specific transcripts and activate transcripts involved in stress pathways, specifically the unfolded protein response (UPR), which can respond to ER stress, and AMP-activated protein kinase (AMPK), an energy stress regulator that responds to ATP depletion. Therefore, to address this potential pathological mechanism for LHON, we used the Complex I inhibitor, rotenone, to simulate the LHON mutations in oligodendroglial cells in order to examine the consequences on both the UPR (PERK→eIF2α→ATF4→CHOP) and AMPK (↓ATP→AMPK→eIF2α) stress pathways.
Western blot analyses were performed to examine the protein expression of the phosphorylated and activated forms of the UPR sensor, PERK, and its downstream target, eIF2α, as well as AMPK in rotenone-treated human oligodendroglial (HOG) cells. The density (intensity/mm2) of the protein bands was measured using the Quantity One software (Bio-Rad). HOG cells treated with increasing concentrations of rotenone were used to measure the transcript levels of ATF4 and CHOP, the main elements in the activation of the UPR, by quantitative RT-PCR. ATP levels and ATP synthesis were measured by a luminometric assay in HOG cells treated with increasing concentrations of rotenone.
Complex I inhibition by rotenone significantly activated the components of the UPR pathway by inducing PERK phosphorylation (p<0.05) and a dose-dependent phosphorylation of eIF2α (p<0.05 to p<0.005) as well as the transcriptional activation of the eIF2α targets, ATF4 (p<0.05) and CHOP (p<0.05 to p<0.005). In addition, we also observed a dose-dependent increase in phosphorylated AMPK protein expression (p<0.05 to p<0.01), which corresponded to a rotenone-dependent decrease in both ATP synthesis (p<0.05 to p<0.005) and ATP levels (p<0.05 to p<0.005).
These results demonstrate that mitochondrial inhibition by rotenone, which produces a similar Complex I deficiency as the LHON mutations, activates both the UPR and AMPK stress pathways in oligodendroglial cells. We suggest that these mechanisms may be relevant to the demyelination and degeneration of the optic nerve observed in LHON.
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