Abstract
Certain missense mutations in optineurin/OPTN and amplification of TBK1 are associated with normal tension glaucoma. A glaucoma-associated variant of OPTN, M98K, induces autophagic degradation of transferrin receptor (TFRC) and death in retinal cells. Here, we have explored the role of Tbk1 in M98K-OPTN-induced autophagy and cell death, and the effect of Tbk1 overexpression in retinal cells. Cell death induced by M98K-OPTN was dependent on Tbk1 as seen by the effect of Tbk1 knockdown and blocking of Tbk1 activity by a chemical inhibitor. Inhibition of Tbk1 also restores M98K-OPTN-induced transferrin receptor degradation. M98K-OPTN-induced autophagosome formation, autophagy and cell death were dependent on its phosphorylation at S177 by Tbk1. Knockdown of OPTN reduced starvation-induced autophagosome formation. M98K-OPTN expressing cells showed higher levels of Tbk1 activation and enhanced phosphorylation at Ser177 compared to WT-OPTN expressing cells. M98K-OPTN-induced activation of Tbk1 and its ability to be phosphorylated better by Tbk1 was dependent on ubiquitin binding. Phosphorylated M98K-OPTN localized specifically to autophagosomes and endogenous Tbk1 showed increased localization to autophagosomes in M98K-OPTN expressing cells. Overexpression of Tbk1 induced cell death and caspase-3 activation that were dependent on its catalytic activity. Tbk1-induced cell death possibly involves autophagy, as shown by the effect of Atg5 knockdown, and requirement of autophagic function of OPTN. Our results show that phosphorylation of Ser177 plays a crucial role in M98K-OPTN-induced autophagosome formation, autophagy flux and retinal cell death. In addition, we provide evidence for cross talk between two glaucoma associated proteins and their inter-dependence to mediate autophagy-dependent cell death.
Highlights
Glaucomas are a complex, heterogeneous and multi-factorial group of neurodegenerative eye diseases, characterized by a progressive degeneration of retinal tissues, retinal ganglion cells
We report that phosphorylation of M98K-OPTN at Ser177 by Tbk1 is required for M98K-OPTN induced cell death, autophagosome formation, autophagy flux and transferrin receptor (TFRC) degradation in retinal cells
Since phosphorylation of M98K at Ser177 is a prerequisite for cell death induced by M98K-OPTN, and phosphorylation on Ser177 potentiates autophagy ability, we further explored whether M98K-OPTN gets phosphorylated better than wild type (WT)-OPTN
Summary
Heterogeneous and multi-factorial group of neurodegenerative eye diseases, characterized by a progressive degeneration of retinal tissues, retinal ganglion cells. It is a major cause of irreversible blindness worldwide. Mutations in OPTN (OPTN) are associated with NTG and account for 1–2% of POAG [10]. Quite a few mutations in OPTN are reported in glaucoma, though only a few mutations, including E50K and M98K have been shown to alter cellular homeostasis and cause degeneration of retinal cells by engagement of distinct mechanisms [13,14,15,16]. The M98K mutation is more prevalent in Asian populations [17,18,19]
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