Glaucoma Glaucoma-Associated Mutations in the Optineurin Gene Have Limited Impact on Parkin-Dependent Mitophagy

Kseniia Chernyshova,1,2 Keiichi Inoue,1 Shun-Ichi Yamashita,1 Takeo Fukuchi,2 and Tomotake Kanki1 1Department of Cellular Physiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 2Department of Ophthalmology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

Correspondence: Shun-Ichi Yamashi- PURPOSE. Glaucoma results in progressive degeneration of the optic nerve and irreversible ta Department of Cellular Physiolo- vision loss. Several mutations in the gene encoding optineurin (OPTN), the receptor for gy, Niigata University Graduate Parkin-dependent mitochondrial autophagy (mitophagy), are associated with glaucoma and School of Medical and Dental Sci- amyotrophic lateral sclerosis (ALS). ALS mutations in the ubiquitin-binding domain of OPTN ences, Niigata 951-8510, Japan; impair Parkin-dependent mitophagy. However, the effects of glaucoma mutations in this [email protected]. Tomotake Kanki, Department of region remain unknown. We examined the impact of glaucoma-associated OPTN mutations Cellular Physiology, Niigata Univer- on Parkin-dependent mitophagy. sity Graduate School of Medical and METHODS. The mitochondria-localized, pH-sensitive fluorescent protein mito-Keima was used Dental Sciences, Niigata 951-8510, to monitor mitophagy. HeLa cells expressing Parkin were treated with carbonyl cyanide 3- Japan; chlorophenylhydrazone (CCCP) or oligomycin/antimycin A (O/A) to induce Parkin-dependent [email protected]. mitophagy. Two complementary mitophagy receptors, OPTN and NDP52, were deleted in KC and KI contributed equally to the HeLa cells expressing mito-Keima and Parkin (DKO_HeLa). The mutant OPTN genes were re- work presented here and should introduced into DKO_HeLa cells using retroviruses or through transfection. Mitophagy therefore be regarded as equivalent activity and OPTN localization were evaluated via microscopic analyses. OPTN binding to authors. ubiquitin was examined using an immunoprecipitation assay. Submitted: March 27, 2019 Accepted: July 14, 2019 RESULTS. Parkin-dependent mitophagy was inhibited in DKO_HeLa cells. Introduction of two glaucoma mutations in the ubiquitin-interacting region of OPTN restored mitophagy in CCCP- Citation: Chernyshova K, Inoue K, treated DKO_HeLa cells, whereas the two ALS mutations failed to replicate this effect. Under Yamashita S-I, Fukuchi T, Kanki T. Glaucoma-associated mutations in the treatment with CCCP, the two glaucoma-mutant OPTN proteins normally translocated to optineurin gene have limited impact mitochondria and bound to ubiquitinated proteins. Furthermore, five additional glaucoma- on Parkin-dependent mitophagy. In- mutant OPTN proteins restored CCCP-induced mitophagy. Moreover, treatment with O/A vest Ophthalmol Vis Sci. exhibited similar results. 2019;60:3625–3635. https://doi.org/ CONCLUSIONS. Glaucoma-mutant OPTN proteins retain their normal properties as mitophagy 10.1167/iovs.19-27184 receptors, suggesting that mutations in the OPTN gene cause glaucoma through a mechanism independent of mitophagy defects. Keywords: glaucoma, mitophagy, mitochondria, optineurin, parkin

laucoma is an eye disease characterized by progressive and the latter is associated with both NTG and juvenile open- G degeneration of the optic nerve, eventually resulting in angle glaucoma.10 The H26D, E50K, and E103D were located in irreversible loss of vision. Glaucoma is a leading cause of the site interacting with TANK binding kinase 1 (TBK1), blindness, and the number of individuals with glaucoma will another causal gene for POAG and amyotrophic lateral sclerosis increase to 79.6 million worldwide by 2020; of those, 11.1 (ALS; Fig. 1B). The T202R mutation was identified in an Indian million will be bilaterally blind.1 The most common form of this patient with POAG.6 Unlike H26D, T202R, and H486R, the disease is POAG, which is associated with elevated IOP. Normal- E50K mutation induces cell death in retinal neuronal cells.11–13 tension glaucoma (NTG), a progressive optic neuropathy with The A336G and A377T mutations were discovered in German normal IOP, is a major subtype of POAG. OPTN, the gene patients with NTG.8 The A377T and H486R mutations were encoding optineurin (OPTN), was the first gene to be located in the ubiquitin-interacting region of the OPTN protein discovered in which mutations are associated with NTG.2 (Fig. 1B). These include missense mutations, such as H26D,3,4 E50K,2 Furthermore, more than 20 mutations in the OPTN gene E103D,5 T202R,6 A336G,7 A377T,8 and H486R5,9 (Fig. 1A). The have been reported as causal mutations leading to ALS, a fatal H26D mutation was first identified in Japanese patients with neurologic disorder accompanied by degeneration of both POAG.4 The association of H26D with POAG was confirmed in upper motor neurons in the motor cortex of the brain and another Japanese patient.3 The E50K mutation was reported in lower motor neurons in the brainstem and spinal cord.14 These adult-onset POAG families together with three other mutations OPTN mutations are present in both familial and sporadic forms (the 691_692insAG insertional frameshift mutation and the of ALS.14 A total of more than 40 mutations have been reported R545Q and M98K missense mutations).2 The mutations E103D for POAG and ALS, and are located throughout the OPTN gene and H486R were identified in Chinese patients with POAG,5 (Fig. 1A). All OPTN mutations are generally segregated by the

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FIGURE 1. Structure of human OPTN and the pathogenic mutations analyzed in this study. (A) Glaucoma- (red) and ALS- (blue) associated mutations in this study and domain structure of OPTN. CC, coiled coil; LZ, leucine zipper; UBD, ubiquitin-binding domain; ZF, zinc finger. (B) Protein interacting regions of OPTN. Two glaucoma- (A377T and H486R) and two ALS- (Q454E and E478G) associated mutations are located in the ubiquitin- interacting region of OPTN.

type of disease. Certain POAG mutations, including R545Q and METHODS 691_692insAG, have been associated with the ALS pheno- type.2,15–17 However, their associations are currently contro- Antibodies versial.3,4,10,15–24 OPTN is a multifunctional protein involved in diverse The primary antibodies used in this study were anti-OPTN cellular processes, including the inflammatory response, (ab23666; Abcam, Cambridge, UK), anti-NDP52 (#9036; Cell maintenance of the Golgi apparatus, vesicle trafficking, and Signaling Technologies, Danvers, MA, USA), anti-hemagglutinin 14,25,26 (anti-HA; M180-3; MBL, Nagoya, Japan), anti-Keima_Red (M126- signal transduction. It has been identified as a primary 3; MBL), anti-green fluorescent protein (anti-GFP; #598; MBL), receptor for selective mitochondrial autophagy, also termed anti-multiubiquitin (D058-3; MBL), and anti-actin (sc-8432; mitophagy.27–29 Mitophagy is a cellular homeostatic process SantaCruz, Dallas, TX, USA). The secondary antibodies used that selectively eliminates damaged mitochondria.30,31 The were horseradish peroxidase (HRP)-conjugated anti-mouse IgG following two distinct mitophagy pathways have been and HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch, identified: the PTEN-induced kinase 1 (PINK1)/Parkin-depen- West Grove, PA, USA). dent and -independent pathways. Both PINK1 and Parkin have been identified as genes responsible for the autosomal recessive form of familial Parkinson’s disease. PINK1 and Plasmid Construction and Mutagenesis 30 Parkin cooperate to execute mitophagy. Newly synthesized The wild-type (WT) OPTN coding sequence from a HeLa cell PINK1 is targeted to the mitochondria, subsequently released cDNA library was amplified through PCR and ligated into a from intact mitochondria, and immediately degraded by the pcDNA3.1-EGFP/Zeo vector to generate an OPTN expression ubiquitin–proteasome system in the cytosol. Following the plasmid. For the introduction of point mutations in the depolarization of mitochondria, PINK1 can accumulate on the pcDNA3.1-EGFP_OPTN(WT) vector, the following primers mitochondrial outer membrane and recruit the ubiquitin ligase were used: H26D; 50-GGA AAT GGA CCC GAC CTG GCC Parkin to the mitochondria. Subsequently, mitochondrial CAC CCA AAC-30, E50K; 50-AAA GAG CTC CTG ACC AAG AAC proteins are ubiquitinated by Parkin. Autophagy receptor CAC CAG CTG-30, E103D; 50-GGC CTT GAG TCA TGA CAA proteins, including OPTN, NDP52, p62, NBR1, and TAX1BP1, TGA GAA ATT GAA G-30, T202R; 50-ATA GTC CTG GGC CCA are translocated to the damaged mitochondria via binding to GGA GAA CAG TCT CCA C-30, A336G; 50-CAA GAA AAG TGT ubiquitinated mitochondrial proteins and direct interactions CAG GGC CTT GAA AGG AAA AAT TCT G-30, A377T; 50-ATC with microtubule-associated protein 1 light chain 3 (LC3) to AAA ATG GAA CAG ACT AAA ACA GAG GAT G-30, Q454E; 50- link damaged mitochondria with autophagosome formation. GCA AAC CAT TGC CAA GGA AGA GGA CCT GG-30, E478G; 50- Mitochondria enveloped in autophagosomes are be delivered CTG ATT TTC ATG CTG GAA GAG CAG CGA GAG-30, and to lysosomes (autolysosomes) and degraded. OPTN is not H486R; 50-CGA GAG AGA AAA TTC GTG AGG AAA AGG AGC- required for the execution of PINK1/Parkin-independent 30. Mutations were introduced via PCR-based mutagenesis mitophagy. using KOD PLUS Neo DNA Polymerase (TOYOBO, Osaka, The OPTN E478G mutation linked to sporadic ALS disrupts Japan) and Dpn I digestion (New England Biolabs, Ipswich, the ubiquitin-binding function of OPTN and affects Parkin- MA, USA). mediated mitophagy.27,29,32 However, it remains unclear whether glaucoma-associated OPTN mutations in the ubiqui- Cell Culture tin-interacting region affect Parkin-mediated mitophagy. In the present study, we hypothesized that mutant OPTN causes Cells were cultured in Dulbecco’s modified Eagle’s medium defects in the Parkin-dependent mitophagy pathway, eventual- (DMEM; #043-30085; Wako Pure Chemical Industries, Osaka, ly leading to POAG. We found that glaucoma-associated OPTN Japan) supplemented with 10% fetal bovine serum (FBS; mutants rescued the mitophagy defect in mitophagy receptor- #10270; Life Technologies, Waltham, MA, USA), and main- deficient HeLa cells (OPTN- and NDP52-deficient cells), tained at 378C under 5% carbon dioxide. For Parkin-dependent whereas ALS-associated OPTN mutants (Q454E and mitophagy, cells were cultured for 3 hours in DMEM/FBS E478G)32,33 did not. containing 10 lM carbonyl cyanide 3-chlorophenylhydrazone

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(CCCP; #034-16993; Wako Pure Chemical Industries), or for 6 subjected to immunoprecipitation, and subsequently incubat- hours in DMEM/FBS containing 10 lM oligomycin (O4533; ed with Dynabeads Protein G (#10003D; Invitrogen) conjugat- Wako Pure Chemical Industries), and 4 lM antimycin A (#514- ed with anti-GFP antibody (#598; MBL) for 30 minutes at room 55521; Wako Pure Chemical Industries). temperature. After washing in PBS containing 0.3% Tween20, the immunoprecipitated samples were eluted in 13 SDS Cell Lines and Mitophagy Assay sample buffer (50 mM Tris-HCl [pH6.8], 2% SDS, 6% b- mercaptoethanol, 10% glycerol) at 958C for 5 minutes, and The HeLa cell line stably expressing HA-Parkin was used as subjected to SDS-PAGE and Western blotting analysis. The blot 34 previously described. The HeLa cell line stably expressing the was probed with anti-GFP antibody or anti-ubiquitin antibody, mitochondria-targeting Keima (mito-Keima) and HA-Parkin was and visualized using EzWestLumi plus (#2332638; ATTO, 35 used as previously described. We performed the mito-Keima Tokyo, Japan). assay to monitor mitophagy.36–38 Keima is a pH-sensitive fluorescent protein. When Keima is present in a neutral Statistics environment (i.e., mitochondrial matrix), this fluorescent protein is excited by 440 nm light (shown in green), but not Statistical analyses were performed using the GraphPad Prism by 590 nm light. However, when Keima is present in acidic 8.1.2 software (GraphPad Software, La Jolla, CA, USA). Values conditions, (i.e., autolysosomes), this fluorescent protein is are expressed as mean 6 SEM. Statistical differences were excited by 590 nm light (shown in red), but not by 440 nm tested using the Kruskal–Wallis test with Dunn’s multiple light (Figs. 2A, 2B). Accordingly, the mitophagy activity was comparisons. P values < 0.05 were considered as statistically estimated by counting the number of autolysosomal punctate significant. structures observed when excited by 590-nm wavelength using the MetaMorph 7 software (Molecular Devices, San Jose, CA, USA). RESULTS

Generation of OPTN/NDP52 Double Knockout Establishment of OPTN/NDP52 DKO HeLa Cells (DKO) HeLa Cells We used the mitochondria-targeting pH-sensitive fluorescent protein mito-Keima to monitor mitophagy (Fig. 2A).36–38,40 We We applied the CRISPR-Cas9 system to HeLa cell lines treated HeLa cells stably expressing mito-Keima and HA-Parkin expressing the mito-Keima and HA-Parkin genes as previously (mtKeima/Parkin_HeLa) with 10 lM mitochondrial uncoupler described to generate and DKO cells.37,39 Guide OPTN NDP52 CCCP or dimethyl sulfoxide (DMSO) and observed mitophagy. RNAs selected from exon 4 (50-CAC CGA AAC CTG GAC OPTN In the control DMSO-treated cells, mito-Keima signals visual- ACG TTT ACC C-30) and exon 2 (50-CAC CGC ATT TCA NDP52 ized with wavelengths of 440 nm were localized to the TCC CTC GTC GAA-30) were ligated into the pX330-U6- mitochondrial matrix, showing a tubular mitochondria-like Chimeric_BB-CBh-hSpCas9 plasmid (#42230; Addgene, Water- morphology (Fig. 2B, shown in green). Upon induction of town, MA, USA) (OPTN-CRISPR1 and NDP52-CRISPR1 plas- mitophagy by CCCP, portions of the mitochondria were mids, respectively). HeLa cells stably expressing mito-Keima delivered into acidic lysosomes and mito-Keima signals and HA-Parkin were transfected with OPTN-CRISPR1 and visualized with wavelengths of 590 nm were observed in pcDNA3.1-hygro() plasmids using the FuGENE HD Transfec- punctate structures (Fig. 2B, shown in red). tion Reagent (E2311; Promega, Madison, WI, USA). The Although OPTN and NDP52 are the primary receptors for following day, the culture media were exchanged with Parkin-mediated mitophagy in HeLa cells,41 five mitophagy DMEM/FBS containing 400 lg/mL hygromycin B for selection. receptors have been reported.30 We initially knocked out After 3 days, the cells were cultured in DMEM/FBS without OPTN and NDP52 in mtKeima/Parkin_HeLa cells using the hygromycin B for 2 days, and subsequently re-plated for single CRISPR/Cas9 system to investigate the specific contribution of colony selection. Each colony derived from a single cell was 39 subjected to western blotting analysis to select OPTN–KO OPTN in the Parkin-mediated mitophagy pathway. Concom- cells. Similarly, OPTN–KO mito-Keima/Parkin HeLa cells were itant deletions of OPTN and NDP52, as well as the stable transfected with the NDP52-CRISPR1 plasmid to generate expression of mito-Keima and HA-Parkin, were confirmed OPTN/NDP52_DKO mito-Keima/Parkin HeLa cells. through Western blotting analysis using specific antibodies (Fig. 2C). We treated the DKO_HeLa cells with 10 lM CCCP or DMSO to confirm whether mitophagy activity was impaired in Microscopic Analysis these cells due to loss of the mitophagy receptor proteins Fluorescent microscopic analysis was performed without OPTN and NDP52. As expected, mitophagy was insufficiently fixation using a microscope (IX73 Olympus, Tokyo, Japan) induced in DKO_HeLa cells after treatment with CCCP for 3 with an UPlanSApo 360 oil objective lens (numerical aperture hours, whereas it was sufficiently induced in mtKeima/ of 1.40). Parkin_HeLa cells (Fig. 2D). Further treatment with CCCP- induced a considerable level of mitophagy even in DKO_HeLa cells. In addition, apoptotic cell death was observed after 12 Immunoprecipitation hours of treatment (Supplementary Fig. S1). Accordingly, we OPTN/NDP52_DKO HeLa cells were transiently transfected decided to use treatment with 10 lM CCCP for 3 hours in the with the described plasmids using FuGENE HD Transfection present study for the evaluation of mitophagy activity. Reagent according to the manufacturer’s instructions. After 72 hours, the cells were treated with 10 lM CCCP for 3 hours and Glaucoma-Mutant OPTN Restored CCCP-Induced lysed in RIPA buffer (25 mM Tris-HCl [pH7.6], 150 mM NaCl, Parkin-Dependent Mitophagy 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 13 protease inhibitor cocktail (#04693132001; Roche, When N-terminally GFP-tagged OPTN_WT was exogenously Basel, Switzerland). The samples were sonicated for a few expressed in DKO_HeLa cells, the mitophagy per cell was seconds, incubated on ice for 30 minutes, and centrifuged at significantly increased after treatment with CCCP for 3 hours. 14,000g at 48C for 15 minutes. The supernatants were In contrast, the expression of GFP alone did not induce

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FIGURE 2. Generation of OPTN and NDP52 double KO HeLa cell line. (A) Schematic of the mito-Keima system. The fluorescent mito-Keima protein is localized to the mitochondrial matrix (‘‘mitochondria’’), and exhibits pH-dependent excitation. The excitation peak (‘‘EX’’) of the mito-Keima protein shifts from 440 to 590 nm when mitochondria are delivered to acidic lysosomes (‘‘mitophagy’’). (B) WT and OPTN/NDP52 DKO_HeLa cells were treated with 10 lM CCCP for 3 hours, and analyzed through immunofluorescence microscopy. The mito-Keima signals transported to lysosomes were considered to represent ‘‘mitophagy’’ (red), and nondegraded mitochondria were designated as ‘‘mitochondria’’ (green). Scale bar: 10 lm. (C) WT and OPTN/NDP52 DKO_HeLa cells were confirmed through western blotting with anti-OPTN, anti-NDP52, anti-Keima, anti-HA, and anti-actin antibodies. (D) Quantification of CCCP-induced mitophagy in WT and DKO_HeLa cells. After treatment with CCCP for 3, 6, or 12 hours, mitophagy signals (shown in red in [B]) per cell were quantified. Values are expressed as mean 6 SEM. White, WT; Black,DKO.

mitophagy (Figs. 3A, 3B). This suggests that WT OPTN is showed a significant, yet milder, reduction in the restored sufficient to restore mitophagy in DKO_HeLa cells. The OPTN level of mitophagy relative to that observed with OPTN_WT. In gene harboring a glaucoma or ALS mutation was expressed in contrast with ALS-associated mutants, the two glaucoma- DKO_HeLa cells to investigate whether disease-associated associated OPTN mutations tested (A377T and H486R) did mutations in this gene can restore Parkin-mediated mitophagy. not alter mitophagy activity relative to OPTN_WT. We tested four disease-associated mutations in the ubiquitin- interacting region of OPTN, which are essential for Parkin- mediated mitophagy,14 including two glaucoma mutations5,8,9 Glaucoma-Mutant OPTN Normally Localized to (A377T and H486R) and two ALS mutations32,33 (Q454E and Mitochondria and Bound to Ubiquitinated Proteins E478G) (Figs. 1A and 3). The E478G mutation of the OPTN gene was least effective in restoring the level of mitophagy When Parkin-mediated mitophagy is induced, mitochondrial among all mutations analyzed. This result is consistent with outer membrane proteins are ubiquitinated by Parkin and 30,41 that reported in a previous publication, showing that ALS- interact with OPTN via ubiquitin binding. Subsequently, associated E478G mutant OPTN was defective in ubiquitin- OPTN interacts with LC3 to cause selective enveloping of binding and autophagosome formation.27,29 Cells with another mitochondria by the autophagosome (Fig. 1B).30,41 Thus, mutant OPTN harboring the ALS-associated Q454E also OPTN accumulates in mitochondria during Parkin-mediated

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FIGURE 3. Glaucoma-mutant OPTN restored Parkin-dependent mitophagy in DKO_HeLa cells. (A) Representative pictures of mito-Keima signals observed in OPTN-transduced DKO_HeLa cells. DKO_HeLa cells were transduced with GFP alone or GFP-OPTN and treated with 10 lM CCCP for 3 hours. The mito-Keima signals transported to the lysosomes were considered to represent ‘‘mitophagy’’ (red), and nondegraded mitochondria were designated as ‘‘mitochondria’’ (green). Scale bars:10lm. (B) Quantification of mitophagy signals shown in (A). The levels of mitophagy were decreased in cells carrying ALS-mutant OPTN proteins (Q454E and E478G) relative to OPTN_WT; however, they were unaltered in cells carrying glaucoma-mutant OPTN proteins (A377T and H486R). Values are expressed as mean 6 SEM. *P < 0.05, **P < 0.01. White, treatment with DMSO; Black, treatment with CCCP.

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FIGURE 4. Glaucoma-mutant OPTN normally localized to mitochondria and bound to ubiquitinated proteins. (A) Representative pictures of GFP- OPTN signals observed in OPTN-transduced DKO_HeLa cells. DKO_HeLa cells were transduced with GFP alone or GFP-OPTN and treated with 10 lM CCCP for 3 hours. The subcellular localizations of exogenous OPTN were examined according to the GFP signals. Scale bars:10lm. (B) Quantification of GFP-OPTN localization shown in (A). The mitochondrial localization was diminished in cells carrying ALS-mutant OPTN proteins (Q454E and E478G) relative to OPTN_WT, but was normal in cells carrying glaucoma-mutant OPTN proteins (A377T and H486R). (C) Representative western blot of the immunoprecipitation assay. DKO_HeLa cells were transfected with GFP-OPTN and treated with 10 lM CCCP for 3 hours. The immunoprecipitated samples from protein lysates with anti-GFP antibody were probed with anti-ubiquitin antibody. Glaucoma-mutant OPTN proteins (A377T and H486R) bound to ubiquitinated proteins (poly-Ub) as OPTN_WT, whereas ALS-mutant OPTN proteins (Q454E and E478G) did not.

mitophagy.41 We examined whether a mutation in the UBD of localized GFP-positive signals were scarcely observed in OPTN alters the localization of OPTN. As expected, GFP- DKO_HeLa cells harboring OPTN_E478G; the majority of the OPTN_WT efficiently co-localized with mitochondria after the GFP-positive signals were diffused in the cytoplasm. The induction of mitophagy by CCCP (Figs. 4A, 4B). The ALS- OPTN_Q454E protein localized both to the mitochondria and associated OPTN_Q454E and _E478G proteins localized the cytoplasm. Thus, the two ALS-associated mutations differently during the induction of mitophagy. Mitochondria- inhibited the translocation of OPTN to the mitochondria.

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Meanwhile, the glaucoma-associated OPTN_A377T and We further examined the binding activity of mutant OPTN OPTN_H486R mutants localized normally to mitochondria proteins to ubiquitinated proteins in the context of O/A- after treatment with CCCP. In summary, glaucoma-associated induced mitophagy by immunoprecipitation. Seven glaucoma- mutant OPTN proteins can still translocate into mitochondria associated mutant OPTN proteins and OPTN_WT were and induce mitophagy upon treatment with CCCP, whereas associated with ubiquitinated proteins that appeared in bands ALS-associated mutant OPTN proteins fail to translocate. These more than 100 kDa (Fig. 6D). Surprisingly, the OPTN_E50K results suggest that glaucoma-associated mutations in OPTN mutant bound more to ubiquitinated proteins than the others. have a markedly limited impact on CCCP-induced mitophagy As expected, the two ALS-associated mutant OPTN proteins compared with ALS-associated mutations. did not fully bind to ubiquitinated proteins (Fig. 6D). We further examined the binding activity of mutant OPTN protein to ubiquitinated proteins in the context of CCCP- induced mitophagy through immunoprecipitation. We pre- DISCUSSION pared protein lysates extracted from CCCP-treated GFP-OPTN The mechanism through which mutations in the OPTN gene transfected cells and immunoprecipitated GFP-OPTN from lead to glaucoma or ALS has not been clarified, although there them with an anti-GFP antibody. The immunoprecipitated is evidence that OPTN plays crucial roles in Parkin-dependent samples were probed with an anti-ubiquitin antibody. The two mitophagy.30,27,41 In the present study, we investigated the glaucoma-associated mutant OPTN proteins and the WT OPTN impact of glaucoma-associated mutations in the OPTN gene in were associated with ubiquitinated proteins that appeared in the context of Parkin-dependent mitophagy. Although these bands more than 100 kDa. As expected, the two ALS-associated mutations had a limited effect on mitophagy regardless of the mutant OPTN proteins did not fully bind to ubiquitinated mutation site, two ALS-associated mutations in the ubiquitin- proteins (Fig. 4C). interacting region of OPTN exerted a more pronounced effect, as previously reported.41 Five Additional Glaucoma-Mutant OPTN Proteins In the present study, we used HeLa cells to study the role Restored Mitophagy and Localized to Mitochondria of OPTN mutants in the context of Parkin-mediated mitoph- agy. This cell line has been widely used for the mechanistic We extended our analysis to other glaucoma-associated study of mitophagy. 34,41,43 The HeLa cell line is an mutations located outside the ubiquitin-interacting region of immortalized cell line derived from cervical carcinoma tissue the OPTN gene. We selected five additional glaucoma- 3,4 2 lacking endogenous expression of Parkin due to gene associated mutations throughout the gene (H26D, E50K, 43–45 5 6 7 truncation. We used HeLa cells stably expressing Parkin E103D, T202R, and A336G ) (Fig. 1A). However, none of the to analyze Parkin-dependent mitophagy.34,43 Constitutive glaucoma-associated mutant OPTN proteins impaired Parkin- overexpression of Parkin may induce a larger magnitude of mediated mitophagy, mitochondrial translocation, or ubiquitin- mitophagy even in the context of OPTN/NDP52 deficiency, binding activity under treatment with CCCP (Figs. 5A–C). which would obscure the impaired mitophagy of glaucoma- Surprisingly, the ubiquitin-binding activity of the OPTN_E50K associated OPTN mutants. We confirmed the previously protein was increased, despite normal mitophagy activity (Fig. reported functional deficits of ALS-associated mutants in this 5C). cell line.27,41 The use of other cell types (e.g., neuronal cells) would be more suitable as the optic nerve is degenerated in Glaucoma-Mutant OPTN Also Restored the patients with glaucoma.1,12 Shim et al.46 reported that acute Mitophagy Induced by the Concomitant Treatment overexpression of the OPTN_E50K mutant in rat primary of Oligomycin and Antimycin A retinal ganglion cells (RGCs) caused increased mitophagy activity as well as activation of the apoptotic Bax pathway and Finally, we examined the impact of mutant OPTN on increased oxidative stress in vitro. The results of our oligomycin and antimycin A (O/A)-induced Parkin-dependent ubiquitin-binding assay using the OPTN_E50K mutant sup- mitophagy, as the effect of CCCP is detrimental for cellular port these findings. Increased binding between OPTN and processes other than mitochondrial integrity. Treatment with ubiquitinated proteins enhances mitophagy activity, despite O/A inhibits the mitochondrial respiratory complex III, leading the absence of alteration in restored mitophagy level to mitochondrial depolarization and increased levels of reactive demonstrated by our mitophagy assay. This discrepancy may oxygen species. The induction of Parkin-dependent mitophagy be attributed to the cell type; RGCs are postmitotic neuronal by treatment with O/A is generally considered more physio- cells, in which the introduced genes are undiluted, whereas logical than that observed through treatment with CCCP.42 HeLa cells are immortalized tumor cells, in which the We treated the DKO_HeLa cells with O/A (10 lM/4 lM) or introduced genes are diluted during cell division. Although DMSO to examine the impairment of mitophagy activity in this was beyond the scope of the present study, it would be these cells. Mitophagy was insufficiently induced in DKO_HeLa interesting to analyze neuronal cells differentiated from cells after treatment with O/A for 6 hours (Fig. 6A and pluripotent stem cells with specific OPTN mutations.47 Supplementary Fig. S3). In DKO_HeLa cells, exogenously Duplication of the TBK1 gene was recently identified in expressed OPTN_WT proteins restored O/A-induced mitoph- patients with NTG.48,49 Both TBK1 and OPTN have functions agy and translocated normally to mitochondria (Fig. 6B and in the autophagy machinery and nuclear factor-jB (NF-jB) Supplementary Fig. S4A). The OPTN_E478G mutant was the signaling. Moreover, TBK1 phosphorylates and activates OPTN least effective in restoring the level of mitophagy and to promote autophagosome formation (Fig. 1B).50 Autophagy translocating to mitochondria after treatment with O/A. activity is enhanced in induced pluripotent stem cell–derived Meanwhile, seven glaucoma-associated OPTN mutations re- retinal cells from NTG patients harboring the TBK1 duplica- stored mitophagy activity like OPTN_WT, and translocated tion.51 Furthermore, TBK1 hemizygous transgenic mice have mainly into mitochondria (Fig. 6C and Supplementary Fig. progressive loss of RGCs. Aged OPTN_E50K transgenic mice S4B). These results suggest that glaucoma-associated mutations also have degeneration of RGCs and elevated mitophagy.46 in OPTN have a markedly limited impact on the restoration of Collectively, mild and persistent activation of mitophagy O/A-induced mitophagy, unlike ALS-associated mutations. activity may govern the onset of POAG.

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FIGURE 5. Five additional glaucoma-mutant OPTN proteins restored mitophagy, localized to mitochondria, and bound to ubiquitinated proteins. (A) Quantification of mitophagy signals. The levels of mitophagy were unaltered in cells carrying five other glaucoma-mutant OPTN genes (H26D, E50K, E103D, T202R, and A336G). The three samples on the left (, GFP, and WT) and the four samples on the right (A377T, Q454E, E478G, and H486R) are shown as the reference (see Fig. 3B), as all 12 samples were analyzed together in the original experiment. Values are expressed as mean 6 SEM. *P < 0.05, **P < 0.01. White, treatment with DMSO; Black, treatment with CCCP. (B) Quantification of GFP-OPTN localization. Mitochondrial localization was normal in cells carrying five other glaucoma-mutant OPTN genes. (C) Representative Western blot of the immunoprecipitation assay. Five additional glaucoma-mutant OPTN proteins bound to ubiquitinated proteins (poly-Ub) as OPTN_WT, whereas the ALS-mutant OPTN (E478G) did not.

Mechanisms other than Parkin-mediated mitophagy may be receptor-interacting kinase 1 (RIPK1) (Fig. 1B).53 OPTN also involved in the pathogenesis of glaucoma.25,52 OPTN is a interacts with cylindromatosis (CYLD) (Fig. 1B), a deubiquiti- multifunctional adaptor protein that mediates a variety of nating enzyme, to cleave polyubiquitin chains from the target cellular processes.25,52 One of the major targets for OPTN is proteins RIPK1 and NEMO, thus preventing the activation of NF-jB signaling, which regulates the expression of many genes NF-jB.54,55 ALS-associated E478G mutant OPTN abolished the involved in the immune response, apoptosis, and the cell inhibition of NF-jB signaling downstream of TNF-receptor cycle.25,52 OPTN is thought to negatively regulate TNFa- activation, as well as the binding to polyubiquitinated proteins induced NF-jB signaling by competing with the NF-jB localized to damaged mitochondria during Parkin-mediated essential modulator (NEMO), an element of the inhibitor of mitophagy.27,32 Moreover, the ALS-associated Q454E mutant the NF-jB kinase complex, for binding to ubiquitinated OPTN exerts partial inhibitory effects on NF-jB signaling.56

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FIGURE 6. Glaucoma-mutant OPTN proteins restored mitophagy and localized to mitochondria. (A) Quantification of O/A-induced mitophagy in DKO_HeLa cells. After treatment with O/A for 3, 6, or 12 hours, mitophagy signals (shown in red in Supplementary Fig. S3) per cell were quantified. Values are were expressed as mean 6 SEM. White, WT; Black,DKO.(B) Quantification of mitophagy signals. The levels of mitophagy were unaltered in cells carrying seven glaucoma-mutant OPTN genes (H26D, E50K, E103D, T202R, A336G, A377T, and H486R). Values are expressed as mean 6 SEM. *P < 0.05, **P < 0.01. White, treatment with DMSO; Black, treatment with O/A. (C) Quantification of GFP-OPTN localization. Mitochondrial localization was normal in cells carrying seven glaucoma-mutant OPTN genes. (D) Representative Western blot of the immunoprecipitation assay. Seven glaucoma-mutant OPTN proteins bound to ubiquitinated proteins (poly-Ub) as OPTN_WT, whereas the ALS- mutant OPTN proteins (Q454E and E478G) did not.

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Our analyses revealed that the Q454E mutant OPTN showed 6. Kumar A, Basavaraj MG, Gupta SK, et al. Role of CYP1B1, partial impairment in Parkin-dependent mitophagy as well. MYOC, OPTN, and OPTC genes in adult-onset primary open- Thus, ALS-associated mutations in the ubiquitin-interacting angle glaucoma: predominance of CYP1B1 mutations in region of OPTN appear to cause dysregulations of both NF-jB Indian patients. Mol Vis. 2007;13:667–676. signaling and mitophagy. 7. Weisschuh N, Alavi MV, Bonin M, Wissinger B. Identification Similar to the ALS-associated mutant OPTN proteins, of genes that are linked with optineurin expression using a glaucoma-associated H486R mutant OPTN causes loss of combined RNAi-microarray approach. Exp Eye Res. 2007;85: interaction between OPTN and cylindromatosis, resulting in 450–461. increased NF-jB signaling induced by inflammatory cytokines 8. Weisschuh N, Neumann D, Wolf C, Wissinger B, Gramer E. (i.e., TNFa, IL-1b, and lipopolysaccharide).54,57 In the TNFa- Prevalence of myocilin and optineurin sequence variants in induced NF-jB signaling pathway, H486R mutant OPTN cannot German normal tension glaucoma patients. Mol Vis. 2005;11: bind sufficiently to ubiquitinated RIPK1, leading to the 284–287. hyperactivation of NF-jB signaling.54,57 However, H486R 9. Chalasani ML, Swarup G, Balasubramanian D. Optineurin and mutant OPTN can interact with ubiquitinated mitochondrial its mutants: molecules associated with some forms of outer membrane proteins in the context of the CCCP-induced glaucoma. Ophthalmic Res. 2009;42:176–184. Parkin-mediated mitophagy pathway. These discrepancies in 10. Willoughby CE, Chan LLY, Herd S, et al. Defining the the ubiquitin-binding capacity may be attributed to the context pathogenicity of optineurin in juvenile open-angle glaucoma. in which OPTN is recruited. It would be interesting to identify Invest Ophthalmol Vis Sci. 2004;45:3122–3130. factors influencing the ubiquitin-binding activity of OPTN. 11. Sirohi K, Kumari A, Radha V, Swarup G. A glaucoma-associated Furthermore, OPTN interacts with Rab8,58 myosin VI,59,60 variant of optineurin, M98K, activates Tbk1 to enhance and transferrin receptors,61 and is thought to play a role in autophagosome formation and retinal cell death dependent intracellular trafficking (Fig. 1B). E50K mutant OPTN potenti- on Ser177 phosphorylation of optineurin. PLoS One. 2015;10: ates Golgi fragmentation and foci formation, and perturbs the e0138289. interaction between OPTN and Rab8 (a critical regulator of 12. Sayyad Z, Sirohi K, Radha V, Swarup G. 661W is a retinal trafficking), leading to neuronal degeneration.62,63 OPTN ganglion precursor-like cell line in which glaucoma-associated carrying an E50K mutation interacts more with transferrin optineurin mutants induce cell death selectively. Sci Rep. receptors than OPTN_WT, and impairs transferrin uptake.61 2017;7:16855. M98K mutant OPTN enhances the interaction of OPTN with 13. Chalasani ML, Radha V, Gupta V, Agarwal N, Balasubramanian Rab12, a GTPase involved in the trafficking and lysosomal D, Swarup G. A glaucoma-associated mutant of optineurin degradation of transferrin receptors.11 It is suggested that selectively induces death of retinal ganglion cells which is inhibited by antioxidants. . 2007; defective protein trafficking may be the cause of glaucoma in Invest Ophthalmol Vis Sci 48:1607–1614. patients with OPTN mutations. Based on the present and previous data, we conclude that various mechanisms are 14. Weil R, Laplantine E, Curic S, Genin´ P. Role of optineurin in the mitochondrial dysfunction: potential implications in involved in the development of glaucoma and ALS. Defects in neurodegenerative diseases and cancer. Front Immunol. 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