BackgroundOptineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process.ResultsWe show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking.ConclusionsOur results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.

The E50K mutation of optineurin acquired the ability to induce cell death selectively in retinal ganglion cells. This cell death was mediated by oxidative stress. The present findings raise the possibility of antioxidant use for delaying or controlling some forms of glaucoma.

SummaryRab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).