A photoreceptor cell line, 661W, derived from a mouse retinal tumor that expresses several markers of cone photoreceptor cells has been described earlier. However, these cells can be differentiated into neuronal cells. Here, we report that this cell line expressed certain markers specific to retinal ganglion cells such as Rbpms, Brn3b (Pou4f2), Brn3c (Pou4f3), Thy1 and γ-synuclein (Sncg), and some other markers of neuronal cells (beta-III tubulin, NeuN and MAP2). These cells also expressed Opn1mw, a cone-specific marker and nestin, a marker for neural precursor cells. Two glaucoma-associated mutants of OPTN, E50K and M98K, but not an amyotrophic lateral sclerosis-associated mutant, E478G, induced cell death selectively in 661W cells. However, in a motor neuron cell line, NSC34, E478G mutant of OPTN but not E50K and M98K induced cell death. We conclude that 661W is a retinal ganglion precursor-like cell line, which shows properties of both retinal ganglion and photoreceptor cells. We suggest that these cells could be utilized for exploring the mechanisms of cell death induction and cytoprotection relevant for glaucoma pathogenesis. RGC-5 cell line which probably arose from 661W cells showed expression of essentially the same markers of retinal ganglion cells and neuronal cells as seen in 661W cells.

Optineurin (OPTN) is an adaptor protein that is involved in mediating a variety of cellular processes such as signaling, vesicle trafficking, and autophagy. Certain mutations in OPTN (gene OPTN) are associated with primary open angle glaucoma, a leading cause of irreversible blindness, and amyotrophic lateral sclerosis, a fatal motor neuron disease. Glaucoma-associated mutations of OPTN are mostly missense mutations. OPTN mediates its functions by interacting with various proteins and altered interactions of OPTN mutants with various proteins primarily contribute to functional defects. It interacts with Rab8, myosin VI, Huntigtin, TBC1D17, and transferrin receptor to mediate various membrane vesicle trafficking pathways. It is an autophagy receptor that mediates cargo-selective as well as non-selective autophagy. Glaucoma-associated mutants of OPTN, E50K, and M98K, cause defective vesicle trafficking, autophagy, and signaling that contribute to death of retinal ganglion cells (RGCs). Transgenic mice expressing E50K-OPTN show loss of RGCs and persistent reactive gliosis. TBK1 protein kinase, which mediates E50K-OPTN and M98K-OPTN induced cell death, is emerging as a potential drug target. Autoimmunity has been implicated in glaucoma but involvement of OPTN or its mutants in autoimmnity has not been explored. In this review, we highlight the main functions of OPTN and how glaucoma-associated mutants alter these functions. We also discuss some of the controversies, such as the role of OPTN in signaling to transcription factor NF-κB, interferon signaling, and use of RGC-5 cell line as a cell culture model.

Mutations in OPTN are associated with glaucoma, an eye disease, and also with amyotrophic lateral sclerosis (ALS), a motor neuron disease. A 2‐bp insertion in OPTN (691_692insAG or 2bpIns‐OPTN) is associated with both glaucoma and ALS. This mutation results in frame shift after 127 amino acids, giving rise to a protein with C‐terminal aberrant sequence. We have explored the mechanism of induction of cell death by this mutant in a motor neuron cell line, NSC‐34, and also in a retinal cell line, 661W. Compared to wild‐type OPTN, this mutant induced more cell death in NSC‐34 and 661W cells. This mutant localizes predominantly in the nucleus whereas normal OPTN localizes in the cytoplasm. Deletion analysis of 2bpIns‐OPTN showed that the aberrant sequence was not essential for cell death induction. This mutant interacts with TANK‐binding kinase 1 (Tbk1) but not with OPTN and activates Tbk1. This mutant induced ER stress in NSC‐34 cells as seen by induction of C/EBP homologous protein (CHOP) and some other genes. Induction of CHOP, autophagosomal protein LC3‐II and cell death by this mutant were abrogated by Tbk1 knockdown and also by 4‐phenylbutyric acid, that inhibits ER stress. Induction of CHOP and cell death by 2bpIns‐OPTN was autophagy dependent as shown by the effect of Atg5 knockdown. This mutant caused increased formation of LC3‐positive aggregates. Treatment of cells with autophagy inducer rapamycin reduced LC3‐positive aggregates, CHOP and cell death induced by 2bpIns‐OPTN. These results suggest that constitutive activation of Tbk1 by 2bpIns‐OPTN leads to impaired autophagy that results in ER stress and cell death.

ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7. Here, we present cryo-EM structures of human Uba7 in complex with UBE2L6, ISG15 adenylate, and ISG15 thioester intermediate that are poised for catalysis of Uba7-UBE2L6-ISG15 thioester transfer. Our structures reveal a unique overall architecture of the complex compared to structures from the ubiquitin conjugation pathway, particularly with respect to the location of ISG15 thioester intermediate. Our structures also illuminate the molecular basis for Uba7 activities and for its exquisite specificity for ISG15 and UBE2L6. Altogether, our structural, biochemical, and human cell-based data provide significant insights into the functions of Uba7, UBE2L6, and ISG15 in cells.

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