Abstract
Purpose :
The goal of this study is to determine whether the optineurin (OPTN) E50K mutant increases NF-kB p50/Bax protein expression, alters oxidative phosphorylation (OXPHOS) system, and triggers mitophagosome formation in primary retinal ganglion cells (RGCs) overexpressing OPTN E50K mutant in vitro.
Methods :
The OPTN E50K mutant was transduced into primary RGCs using an adeno-associated type 2 viral transduction system in vitro. NF-kB p50 and Bax protein expression were assessed in cultured RGCs by Western blot analysis. Mitochondrial alterations were assessed by transmission EM (TEM) and by Western blot analysis using antibodies for dynamin-related protein 1 (DRP1), phopho-DRP1 at Serine 616 (S616), optic atrophy type 1 (OPA1), OXPHOS complex (Cx), superoxide dismutase 2 (SOD2), peroxisome-proliferator-activated receptor-g-coactivator-1 alpha (PGC-1a) and mitochondrial transcription factor A (Tfam). Mitophagosome formation was assessed by Western blot analysis using a LC3 antibody and by EM tomography. Transport dynamics of axonal mitochondria was assessed by in vitro time-lapse imaging.
Results :
Enhanced OPTN E50K mutation significantly increased the expression levels of NF-kB p50 and Bax, as well as OXPHOS Cx (Cx I, II and IV) protein in cultured RGCs. In contrast, the OPTN E50K mutant sigificanlty decreased the expression levels of OPA1 protein and DRP1 S616 phosphorylation. in cultured RGCs. Quantitative analysis using TEM revealed that the OPTN E50K mutant triggered a significant increase of mitochondrial number, but decreases of mitochondrial lengths and volume density in cultured RGC soma in vitro. In a good agreement with these results, the OPTN E50K mutant siginificantly decreased the expression levels of SOD2, PGC-1a and Tfam protein in cultured RGCs. EM tomography analysis revealed that the OPTN E50K mutant triggered autophagosome and mitophagosome formation that include a well-defined cristae in the degenerating mitochondrion that is enveloped in the autophagosome in cultured RGC soma, as evidenced by increasing LC3-II protein expression. However, the OPTN E50K mutant did not change the transport dynamics of axonal mitochondria in cultured RGCs.
Conclusions :
These results sugget that the OPTN E50K mutant may activate NF-kB p50/Bax pathway, alter mitochondrial OXHPHOS system, and subsequently trigger fission-mediated mitochondrial loss and mitophagosome formation in RGC degeneration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.