Accepted Manuscript Defects in autophagy caused by glaucoma-associated mutations in optineurin Kapil Sirohi, Ghanshyam Swarup PII:

S0014-4835(15)30016-6

DOI:

10.1016/j.exer.2015.08.020

Reference:

YEXER 6757

To appear in:

Experimental Eye Research

Received Date: 25 February 2015 Revised Date:

14 July 2015

Accepted Date: 18 August 2015

Please cite this article as: Sirohi, K., Swarup, G., Defects in autophagy caused by glaucoma-associated mutations in optineurin, Experimental Eye Research (2015), doi: 10.1016/j.exer.2015.08.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Defects in autophagy caused by glaucoma-associated mutations in optineurin Kapil Sirohi and Ghanshyam Swarup* Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research,

*

To whom correspondence should be addressed:

Ghanshyam

Swarup,

Tel.;

+91-40-27192616;

Fax:

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Hyderabad- 500 007, INDIA

+91-40-27160591;

e.mail:

[email protected]; Centre for Cellular and Molecular Biology, Council of Scientific and

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Industrial Research, Hyderabad- 500 007, India.

Keywords: optineurin, autophagy, glaucoma, mutations, E50K-OPTN, M98K-OPTN

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Abbreviations:

ALS, Amyotrophic Lateral Sclerosis; ATG, Autophagy-related proteins; CDK1, Cyclin dependent kinase-1; CMA, Chaperon-mediated autophagy; CYLD, cylindromatosis (turban tumor syndrome) protein; GAP, GTPase activating protein; LC3-1, Microtubule-associated protein 1 light chain 3; NEMO, NF-κB essential modulator; NF-kB, Nuclear factor kappa B; NTG, Normal tension glaucoma; OPTN, Optineurin; POAG, Primary open-angle glaucoma;

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Rab8, Rat sarcoma (abbreviated as Ras)-related protein 8; RGC, Retinal ganglion cells; ROS, Reactive oxygen species; RIP, Receptor interacting protein; TBK1, TRAF family member-associated NF-kappa-B activator kinase 1 (TRAF: Tumor necrosis factor (TNF) receptor-associated factor); TBC1D17, TBC1 domain family member 17; TFRC, Transferrin

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receptor-1; UBD, Ubiquitin-binding domain

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ACCEPTED MANUSCRIPT Abstract Certain mutations in optineurin (gene OPTN) are associated with primary open angle glaucoma. Optineurin is ubiquitously expressed but it shows high level of expression in certain cells and tissues including retinal ganglion cells. It interacts with many proteins, often acting as an adaptor to link two or more proteins. These interactions play a crucial role in

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mediating various functions of optineurin such as membrane vesicle trafficking, autophagy, signal transduction etc. Autophagy is basically a quality control mechanism to remove damaged proteins and organelles through lysosomal degradation. Optineurin was identified as an autophagy receptor that directly interacts with autophagosomal protein, LC3, and

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ubiquitin. These interactions are important for autophagy receptor function. Autophagy receptors recruit their cargo and take it to autophagosomes which fuse with lysosomes to form autolysosomes where degradation of proteins takes place. Optineurin interacts with a

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motor protein, myosinVI, and this interaction is involved in mediating fusion of autophagosomes with lysosomes. A glaucoma-associated mutant of optineurin, E50K, impairs autophagy as well as vesicle trafficking, leading to death of retinal cells by apoptosis. E50K-OPTN-induced block in autophagy is dependent on a GTPase activating protein, TBC1D17. The E50K mutant also causes other changes in the cells such as altered interaction

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with TBK1 protein kinase, aggregate formation, generation of reactive oxygen species and inhibition of proteasome, which may contribute to pathogenesis.

A polymorphism of

optineurin, M98K, associated with glaucoma, causes enhanced autophagy leading to transferrin receptor degradation and apoptotic death of retinal cells. M98K-OPTN-induced

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autophagic cell death is dependent on Rab12 GTPase. Thus, an optimum level of optineurinmediated autophagy is crucial for survival of retinal cells, and impaired autophagy is likely to

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contribute to glaucoma pathogenesis. How impaired autophagy caused by optineurin mutants leads to apoptosis and cell death, is yet to be explored.

1. Introduction

Mutations in the gene OPTN that codes for the protein optineurin are associated with

glaucoma and ALS (amyotrophic lateral sclerosis) (Maruyama et al., 2010; Rezaie et al., 2002). Both of these are neurodegenerative diseases. There are several types of glaucomas. In adults, POAG (primary open angle glaucoma) and angle closure glaucoma are major types of glaucoma. In POAG the increase in intraocular pressure (IOP) is the major risk factor. However, in NTG (normal tension glaucoma), a sub-type of POAG, the intraocular pressure remains in the normal range and it accounts for about 30% of POAG cases (Fingert, 2011).

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ACCEPTED MANUSCRIPT POAG causes permanent bilateral blindness due to progressive loss of retinal ganglion cells in the optic nerve head. Other cells in retina such as photoreceptor cone cells are also lost in POAG (Agudo-Barriuso et al., 2013; Choi et al., 2011; Harada et al., 2007). Rezaie et al. in 2002 identified mutations in the coding region of OPTN that were associated with 16.7% of families affected with autosomal dominant adult onset NTG (Rezaie et al., 2002). One of the

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mutations, E50K, segregated with the disease phenotype in a very large family, providing strong evidence to support the hypothesis that mutations in OPTN cause NTG (Rezaie et al., 2002). Subsequently, several studies reported association of OPTN mutations with NTG in familial as well as sporadic cases of NTG and occasionally with POAG (Alward et al., 2003;

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Ayala-Lugo et al., 2007). Most of the OPTN mutations are missense single copy mutations suggesting, therefore, that these are likely to be dominant. A 2-bp insertion that causes frameshift and truncation was also associated with NTG (Rezaie et al., 2002). In 2010,

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Maruyama et al., showed that certain mutations in OPTN cause ALS, a fatal disease that results from loss of motor neurons in the brainstem, primary cortex and spinal cord leading to paralysis of voluntary muscles (Maruyama et al., 2010). ALS causing mutations in OPTN include deletions, truncations and missense mutations (Fig. 1).

Most of the mutations

associated with ALS are not associated with glaucoma (Fig. 1). On the basis of the nature of

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mutations, it was suggested that loss of function as well as gain of function mechanisms are involved in the pathogenesis of glaucoma and ALS. Optineurin is also seen in pathological structures found in several neurodegenerative diseases including Alzheimer’s disease and

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Parkinson’s disease (Osawa et al., 2011).

2. Structure of optineurin

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Optineurin is mostly a coiled coil protein which does not have any enzymatic activity. Human optineurin is a 577 amino acid protein which migrates as a 74 kDa polypeptide on SDS-PAGE. In the cell optineurin forms hexamers (Ying et al., 2010). Upon induction of oxidative stress in the cell it forms covalent trimers which are not linked by disulfide bonds (Gao et al., 2014). Optineurin has a well defined ubiquitin-binding domain (UBD), zinc finger domain and LC3-interacting region (LIR) (Fig. 1). It interacts directly with several proteins in the cell through well defined binding sites. C-terminal half of optineurin shows homology with NF-κB essential modulator (NEMO or IKKγ), regulatory sub-unit of IKK complex, which is involved in signaling to transcription factor NF-κB (Schwamborn et al., 2000).

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ACCEPTED MANUSCRIPT 3. Sub-cellular localization of optineurin Optineurin is predominantly a cytosolic protein. However, a small fraction of optineurin is present in the Golgi, endocytic vesicles and autophagosomes (Nagabhushana et al., 2010; Park et al., 2010; Rezaie et al., 2002; Wild et al., 2011; Ying and Yue, 2012).

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Overexpressed optineurin forms vesicles some of which are autophagosomes and some are TFRC (transferrin receptor)-positive endosomes (Sirohi et al., 2013). Upon induction of acute

have a defined nuclear localization signal.

4. Interactions and functions of optineurin

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oxidative stress, it migrates to nucleus (De Marco et al., 2006). However, OPTN does not

Optineurin interacting proteins have been identified using several methods such as

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yeast two-hybrid screening, immunoprecipitation and GST pull-down assays (Chalasani et al., 2009; Kachaner et al., 2012b). Nature and function of these interacting proteins provided initial clues about the cellular functions of optineurin. Most of the functions of optineurin are mediated by its ability to link two or more proteins (Fig. 1). UBD of optineurin is involved in most of its functions such as signaling to NF-κB, autophagy and vesicle trafficking. It binds

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to polyubiquitinated proteins with high affinity and shows selectivity towards Lys63-linked and linear polyubiquitin chains (Laplantine et al., 2009). It does not interact with Lys48linked polyubiquitin chains (Zhu et al., 2007). This selectivity is mediated by UBD and zinc finger domain, which also binds to ubiquitin. Mutation of a conserved amino acid Asp474 to

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Asn (D474N) in the UBD abolishes binding of optineurin to ubiquitin.

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5. Optineurin and autophagy

Ubiquitin proteasome system and autophagy are two major pathways for degradation

of proteins in the cell. Macro-autophagy (hereafter referred to as autophagy) is a catabolic mechanism involved in the maintenance of cellular homeostasis by degrading long lived proteins, damaged and aggregated proteins and organelles through lysosomal pathway (Levine and Klionsky, 2004). The cargo that is to be degraded is recruited to a double membrane structure known as autophagosome, which fuses with lysosomes to form autolysosomes where cargo degradation takes place. Autophagy is initiated by activation of Ulk1 protein kinase which is regulated positively by AMP activated protein kinase and negatively by mTORC-1 kinase. Nucleation of autophagy results in the formation of a

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ACCEPTED MANUSCRIPT membranous structure, phagophore (also known as isolation membrane). This phagophore expands and then closes to give rise to autophagosome. During formation of autophagosome, lipidation of LC3-1 occurs, leading to formation of LC3-II which gets incorporated into autophagosomal membrane. LC3-II is considered as a marker for autophagosome. Recruitment of selective cargoes is mediated by autophagy receptors which directly interact

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with autophagosomal protein LC3 and ubiquitin. Optineurin binds to LC3 through its LIR in the N-terminal region. Autophagy receptors such as optineurin, p62, NDP52 etc. mediate cargo selective autophagy by recognizing and binding to the ubiquitinated cargo through UBD (Pankiv et al., 2007; Thurston et al., 2009; Wild et al., 2011). Autophagy receptor

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function of optineurin is regulated by phosphorylation at Ser177 in the LIR which enhances its interaction with LC3. Autophagic function of optineurin is involved in clearance of cytosolic Salmonella and aggregated proteins (Korac et al., 2013; Wild et al., 2011).

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In addition to its function as an autophagy receptor, optineurin also plays a role in maturation of autophagosomes to autolysosomes. Movement of several membrane vesicles, including autophagosome, is mediated by molecular motors that move on the actin cytoskeleton. Optineurin directly interacts with a molecular motor myosin VI, and this interaction recruits myosin VI to the autophagosomes. Binding region of optineurin and other

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autophagy receptors to myosin VI overlaps with UBD (Tumbarello et al., 2012). This myosin VI-optineurin interaction is also involved in trafficking of vesicles between Golgi and plasma membrane (Sahlender et al., 2005).

Role of optineurin as an autophagy receptor for the elimination of damaged

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mitochondria has been shown recently (Wong and Holzbaur, 2014). Mitophagy is a quality control measure of cells to maintain the number of functional mitochondria. Mitochondrial

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dysfunction has been implicated in several neurodegenerative diseases like glaucoma, ALS and Parkinson’s disease. Optineurin is recruited via its UBD to damaged mitochondria which is ubiquitinated by parkin. Subsequently, optineurin recruits LC3 via its LIR for autophagosome formation around damaged mitochondria (Wong and Holzbaur, 2014). ALSassociated optineurin mutant, E478G-OPTN, is defective in formation of autophagosome around damaged mitochondria and this mutation is in the UBD of optineurin. Recently, Lys48-linked ubiquitination of optineurin by tumor-suppressor HACE1, an E3-ubiquitin ligase has been shown in lung cancer cells (Liu et al., 2014). This study sheds light on the importance of the autophagy receptor function of optineurin in suppression of tumors.

6. Non-autophagic functions of optineurin

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ACCEPTED MANUSCRIPT Besides autophagy, optineurin is involved in many functions such as vesicle trafficking and signal transduction. Some of the signalling pathways mediated by optineurin may influence autophagy directly or indirectly.

6.1. Role of optineurin in vesicle trafficking and Golgi maintenance.

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Optineurin interacts with many proteins that are involved in intracellular vesicle trafficking such as Rab8, huntingtin, myosin VI, TFRC and TBC1D17. It was suggested that interaction of optineurin with actin-based motor protein, myosin VI, and a protein involved in microtubule-based motor activities, huntingtin, and the Rab GTPase, Rab8, help in Golgi

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structure maintenance. Optineurin binds to the tail domain of myosin VI through its Cterminal region (Sahlender et al., 2005). Knockdown of optineurin led to loss of myosin VI from Golgi suggesting optineurin’s role in the recruitment of myosin VI to Golgi.

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Optineurin’s interaction with Rab8 and myosin VI plays a role in vesicle fusion at plasma membrane during exocytosis. Optineurin is required for recruitment of myosin VI to Rab8 vesicular structures (Sahlender et al., 2005). Optineurin forms a functional link between Rab8 and myosin VI during basolateral targeting of AP1B dependent cargo in polarized epithelial cells (Au et al., 2007). Through its N-terminal region optineurin interacts and colocalizes

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preferentially with GTP bound form of Rab8 indicating that optineurin is an ‘effector’ of Rab8 (Hattula and Peranen, 2000). Optineurin interacts with TFRC and has a role in its endocytic trafficking to the recycling endosomes. E50K mutant of optineurin impairs the trafficking of TFRC (Nagabhushana et al., 2010; Park et al., 2010). The recycling of TFRC to

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plasma membrane is regulated by Rab8. Optineurin functions as an effector for Rab8 and, in addition, negatively regulates TFRC recycling by recruiting TBC1D17, which directly

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interacts with optineurin. TBC1D17 functions as a GAP for Rab8 and regulates Rab8mediated TFRC recycling (Vaibhava et al., 2012).

6.2. Role of optineurin in NF-κB signalling Nuclear factor kappa B (NF-κB) is an inducible transcription factor, involved in cell

survival and proliferation, immune response and inflammation. Inactive NF-κB is generally sequestered in the cytoplasm by IκB family proteins and upon activation it translocates to the nucleus to induce its target genes (Hayden and Ghosh, 2004). Optineurin acts as a negative regulator in TNFα-induced NF-κB signalling by competing with NEMO for binding to polyubiquitinated RIP (Zhu et al., 2007). Optineurin shows direct interaction with CYLD

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ACCEPTED MANUSCRIPT (Chalasani et al., 2009), a deubiquitinase which deubiquitinates polyubiquitinated RIP. Optineurin recruits CYLD to polyubiquitinated RIP complex, facilitates its deubiquitination and blocks the downstream activation of NF-κB pathway (Nagabhushana et al., 2011). A glaucoma-associated mutant of optineurin, H486R, which is mutated in the UBD, shows reduced interaction with CYLD and is defective in inhibiting TNFα-induced NF-κB

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activation (Nagabhushana et al., 2011). TNF-α induces OPTN gene expression via NF-κB, which has a binding site in OPTN promoter. TNF-α induced optineurin expression in turn dampens TNF-α signalling in a negative feedback loop (Sudhakar et al., 2009). However, recently it has been shown that in immune cells optineurin is dispensable for NF-κB

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activation but is necessary for IRF3 activation (Munitic et al., 2013). Optineurin interacts with UXT (Chalasani et al., 2009), which is involved in TNF-α-induced activation of NF-κB

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target gene promoter. The possible role of optineurin in regulation of UXT function is yet to be elucidated.

6.3. Role of Optineurin in antiviral signalling:

Generally, viral infection stimulates innate immune responses in the host to curb the infection, including production of several proinflammatory cytokines such as type I

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interferons - IFNα/IFNβ, and chemokines (Pietras et al., 2006). However, cytokine signalling is tightly controlled to prevent unwanted tissue damage. Optineurin was shown to act as a negative regulator in antiviral signalling. Infection by Sendai-virus induces expression of optineurin which in complex with TBK1 and ubiquitin ligase TRAF3, inhibits IFNβ

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production as a negative feedback mechanism (Mankouri et al., 2010). However, in response to LPS and double stranded RNA, optineurin positively regulates TBK1-mediated IRF-3

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phosphorylation and IFN-β production in murine bone marrow-derived macrophages (Gleason et al., 2011). The reason for these apparently conflicting reports is not clear. Optineurin interacts with the viral TAX1 oncoprotein which is known to activate NF-

κB signalling mediated through NEMO. Optineurin concurrently binds to TAX1 and TAX1 binding protein 1 (TAX1BP1) and cooperatively increases TAX1 ubiquitination and TAX1mediated NF-κB signalling (Journo et al., 2009).

6.4. Role of optineurin in cell division Optineurin plays an important role in regulating the function of PLK1, a serinethreonine kinase which plays a crucial role in cell cycle progression. Depletion of optineurin

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ACCEPTED MANUSCRIPT induces multinucleation and chromosomal segregation defects. This study suggested the role of optineurin as a negative regulator in mitosis (Kachaner et al., 2012a) .

7. Processing of optineurin Involvement of autophagy and ubiquitin-proteasome system pathways in processing

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of endogenous optineurin has been investigated using inhibitors of these two pathways. Earlier it was reported that endogenous optineurin is degraded primarily through proteasomal pathway in RGC-5, a retinal cell line and PC12, a neuronal cell line (Shen et al., 2011). But subsequent studies have shown that endogenous optineurin is degraded through autophagy in

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RGC-5, HeLa and IMR32 cells whereas overexpressed optineurin is degraded mainly through proteasome (Sirohi et al., 2013). The reason for these apparently conflicting reports is not clear. Degradation of endogenous optineurin by autophagy is consistent with its function as

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an autophagy receptor.

8. The issue of identity of RGC-5 cell line:

Molecular mechanisms of pathogenesis of glaucoma caused by mutations in optineurin have been investigated using cell culture and transgenic mouse models. For this

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purpose RGC-5, a retinal cell line has been used by several groups. RGC-5 cell line, originally described as a rat retinal ganglion cell line, has been re-characterized and found to be of mouse origin with properties/markers of neural precursor cells from retina and it is more likely to be derived from 661W photoreceptor cell line (Krishnamoorthy et al., 2013;

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Van Bergen et al., 2009; Wood et al., 2010). Therefore, it is necessary to re-interpret the data obtained using RGC-5 cells where the papers draw conclusions concerning retinal ganglion

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cell-specific functions.

9. Functional defects caused by OPTN mutants:

9.1. E50K-OPTN impairs autophagy and vesicle trafficking: Effect of overexpression of optineurin mutants in RGC-5 cells, a retinal cell line, has been examined and it was observed that E50K and M98K mutants induced significantly more cell death than wild-type optineurin. Several other mutants such as H486R, H26D, T202R did not induce more cell death than wild-type optineurin (Chalasani et al., 2007; Sirohi et al., 2013). This has been confirmed in transgenic mice expressing E50K-OPTN, which show degeneration of RGCs and thinning of all retinal cell layers (Chi et al., 2010). This is

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ACCEPTED MANUSCRIPT consistent with recent observations suggesting that in human glaucoma, loss of RGCs is accompanied by loss of photoreceptor cone cells (Choi et al., 2011). The E50K mutant forms large foci/vesicle-like structures in the cells which are positive for transferrin receptor, whereas wild-type optineurin forms smaller foci. The E50K mutant inhibits endocytic recycling of TFRC which partly contributes to formation of TFRC-positive foci by E50K-

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OPTN (Nagabhushana et al., 2010; Park et al., 2010). In addition, E50K-OPTN inhibits autophagy which also contributes to formation of large foci. E50K-OPTN-induced death of RGC-5 cells is largely mediated by inhibition of autophagy as shown by the effect of autophagy inducer, rapamycin, which protects against E50K-induced cell death (Chalasani et

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al., 2014; Shen et al., 2011). Block in TFRC recycling also contributes to induction of cell death because co expression of TFRC partially protects retinal cells from E50K-OPTNinduced cell death. Several observations suggest that E50K mutant induces a block in

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autophagy. E50K-OPTN expressing cells show increased level of LC3II and p62, reduced autophagy flux and formation of large LC3-positive foci (Chalasani et al., 2014; Shen et al., 2011).

The E50K mutant was reported to increase autophagy on the basis of increase in LC3-II level

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observed in E50K-expressing RGC-5 cells and in the rat retina upon adeno-virus mediated E50K overexpression (Shen et al., 2011; Ying et al., 2015)). However, increase in LC3 level can also occur if there is a block in autophagy, as interpreted by others (Chalasani et al., 2014). Treatment with rapamycin, an inducer of autophagy, resulted in reduced apoptosis

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induced by the E50K mutant (Shen et al., 2011), suggesting therefore that E50K-induced cell death is mediated by a block in autophagy (Chalasani et al., 2014). Thus, although these two

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groups have observed similar effects of rapamycin on E50K-induced cell death, their interpretations of the effect of E50K mutant on autophagy are different (Chalasani et al., 2014; Shen et al., 2011).

The mechanism of E50K-OPTN-induced block in autophagy has been investigated

recently. Optineurin interacts with TBC1D17, a GAP for Rab8, which inhibits autophagy through its catalytic activity (Chalasani et al., 2014). The E50K-OPTN-induced block in autophagy is mediated by TBC1D17 as shown by the effect of knockdown of TBC1D17 on E50K-induced inhibition of autophagy. Cell death induced by E50K-OPTN is also inhibited by knockdown of TBC1D17 (Chalasani et al., 2014). TBC1D17, through its catalytic activity, mediates E50K-OPTN-induced block in autophagy that leads to death of retinal cells. The

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ACCEPTED MANUSCRIPT mechanism of inhibition of autophagy by TBC1D17 is not clear. Since catalytic activity is required, TBC1D17 is likely to be acting on one of the Rab GTPases involved in autophagy. However, the Rab GTPase that is a substrate of TBC1D17 and is involved in autophagy is yet to be identified. E50K-OPTN-induced inhibition of TFRC recycling is also mediated by TBC1D17 (Vaibhava et al., 2012). E50K mutant impairs TFRC recycling to the plasma

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membrane by enhanced inactivation of Rab8 by TBC1D17. Thus, TBC1D17 mediates E50KOPTN-induced retinal cell death by two different mechanisms, impaired autophagy and TFRC recycling (Chalasani et al., 2014) (Fig. 2). Whether impaired TFRC recycling also

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contributes to impaired autophagy by E50K-OPTN, is yet to be investigated.

The role of Rab8 in impaired TFRC trafficking has been investigated by several groups. Using a fluorescence-based protein complementation assay that measures direct

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protein-protein interaction, Chi et al (2010) reported that the E50K mutant has lost the ability to interact directly with Rab8, whereas others reported that, in immunoprecipitation experiments, Rab8 shows better interaction with E50K mutant as compared to wild type optineurin (Nagabhushana et al., 2010; Park et al., 2010). This altered interaction with Rab8 possibly contributes to impaired protein trafficking by the E50K mutant. Because

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immunoprecipitation experiments cannot distinguish between direct and indirect interaction, this issue of increased or decreased interaction of Rab8 with the E50K mutant could be resolved by the following suggestions (Vaibhava et al., 2012). The E50K mutant and wild type optineurin form a multi-molecular complex in which Rab8, TBC1D17 and perhaps other

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proteins, such as TFRC, are also present. The E50K mutant has lost direct interaction with Rab8 (as seen by yeast two-hybrid assay and also in mammalian cells) but indirect interaction

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through other proteins is enhanced. These altered interactions possibly change the functional positioning of the molecules in the multi-molecular complex in a way that facilitates enhanced inactivation of Rab8 by TBC1D17 in E50K-expresssing cells, leading to impaired TFRC recycling (Vaibhava et al., 2012).

E50K-OPTN expression in retinal cells induces formation of reactive oxygen species (ROS) possibly by mitochondria (Chalasani et al., 2007). Inhibition of ROS by treatment of cells with antioxidant, N-acetyl cysteine, or by overexpression of mitochondrial superoxide dismutase, prevents E50K-OPTN-induced cell death (Chalasani et al., 2007). The oxidative stress generated by E50K-OPTN expression results in formation of covalent trimers of E50KOPTN which is prevented by antioxidants (Gao et al., 2014). Formation of covalent trimers

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ACCEPTED MANUSCRIPT by E50K-OPTN might contribute to retinal cell death seen in glaucoma pathogenesis, but this needs to be examined experimentally. The mechanism by which E50K-OPTN induces ROS production is yet to be investigated. Since optineurin is involved in mitophagy, role of autophagy inhibition in ROS production by E50K-OPTN needs to be investigated.

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E50K-OPTN has been shown to form insoluble aggregates in HEK293 cells and also in neurons derived from induced pluripotent stem cells from a glaucoma patient carrying E50K mutation (Minegishi et al., 2013). A protein kinase, TBK1 has been implicated in these aggregate formation and glaucoma pathogenesis. TBK1 shows direct interaction with OPTN

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and this interaction is enhanced by E50K mutation (Morton et al., 2008). This enhanced interaction is speculated to be involved in aggregate formation by E50K-OPTN and glaucoma pathogenesis (Minegishi et al., 2013). Inhibition of autophagy by E50K-OPTN might also

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contribute to formation of aggregates. Amplification of TBK1 is seen in certain NTG cases although no mutation in this gene is reported. This gene amplification leads to enhanced expression of TBK1 which might contribute to pathogenesis (Awadalla et al., 2015; Fingert et al., 2011). TBK1 is known to phosphorylate optineurin at Ser177 that leads to enhanced interaction with the autophagosomal protein LC3 which mediates autophagy (Wild et al.,

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2011). Whether increased binding of E50K-OPTN with TBK1 results in altered phosphorylation of E50K-OPTN, or some other protein is yet to be investigated. Inhibition of proteasome activity has been observed in E50K-OPTN expressing cells in culture and also in retina of E50K-OPTN transgenic mice (Shen et al., 2011). Proteasome

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function is impaired in some neurodegenerative diseases and it might contribute to aggregate/foci formation. However, the role of proteasome inhibition in E50K-OPTN-

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induced cell death or glaucoma pathogenesis is not clear and requires further investigation.

9.2. M98K-OPTN polymorphism causes enhanced autophagy: The optineurin variant M98K is commonly present in many populations and in the

first study this variant was found to be associated with glaucoma (Rezaie et al., 2002). Subsequent studies have shown that this variant has significant association with glaucoma in Asian populations but not in Caucasian populations (Alward et al., 2003; Ayala-Lugo et al., 2007; Kumar et al., 2007; Sripriya et al., 2006). Functional defects induced by this variant in cell culture system have been shown, where, overexpression of M98K-OPTN induces autophagy and depletes cellular TFRC level by engaging autophagic machinery and causes cell death selectively in retinal cells (Sirohi et al., 2013). Depletion of Atg5 or inhibition of

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ACCEPTED MANUSCRIPT lysosomal activity inhibits M98K-OPTN-induced cell death. TFRC degradation plays a crucial role in M98K-OPTN-induced death of retinal cells because co expression of TFRC or blocking of TFRC degradation inhibits M98K-OPTN-induced cell death. Wild-type optineurin, or another glaucoma-associated mutant E50K, did not induce TFRC degradation suggesting the involvement of different molecular mechanism in cell death induced by E50K

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mutant. Binding to ubiquitin and LC3 is required for M98K-OPTN induced autophagy, TFRC degradation and cell death as mutational inactivation of UBD or LIR inhibits M98KOPTN-mediated autophagy function, suggesting that M98K-OPTN -induced retinal cell death involves autophagic signalling (Sirohi et al., 2013). M98K-OPTN expression potentiates the

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delivery of TFRC to autophagosomes for its degradation in lysosomes which results in the reduced TFRC levels and perturbed cellular homeostasis (Sirohi et al., 2013) (Fig. 3).

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The main function of TFRC is to regulate iron metabolism. Iron in the plasma binds with transferrin, and this iron-transferrin complex interacts with TFRC, a transmembrane receptor on the cell surface. This interaction initiates endocytosis of transferrin-TFRC complex leading to formation of specialized endosomes. Iron is released in the cell due to low pH of the endosomes and transferrin-TFRC complex is recycled back to the plasma

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membrane, either directly from early endosomes (fast recycling) or through recycling endosomes (slow recycling) (Maxfield and McGraw, 2004). A small fraction of TFRC is not recycled and is degraded through autophagy (Sirohi et al., 2013). During autophagy, formation of autophagosomes requires membrane which is contributed by several organelles,

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including TFRC-positive recycling endosomes (Longatti et al., 2012). Whether TFRC contributes to autophagy by facilitating the delivery of membrane from recycling endosomes

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to autophagosomes, or in any other way, is not known.

Reduced TFRC function seems to be the main cause of RGC death induced by M98K-

OPTN because this cell death is prevented by iron supplementation (Sirohi et al., 2013). Impaired regulation of iron metabolism is associated with neurodegeneration in humans and mice (LaVaute et al., 2001; Levy et al., 1999; Rouault and Cooperman, 2006). TFRC deficient mice do not survive beyond 12.5 days and the TFRC deficient embryos display abnormal nervous and hematopoietic systems (Levy et al., 1999). It appears that TFRC has a crucial function in neural and hematopoietic cells. Optineurin directly interacts with TFRC and regulates its endocytic trafficking and recycling (Nagabhushana et al., 2010; Park et al., 2010; Vaibhav et., 2012). Therefore, it is not surprising that an optineurin mutant, M98K,

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ACCEPTED MANUSCRIPT affects TFRC function and perhaps iron metabolism, leading to death of retinal cells (Sirohi et al., 2013). However, further studies are needed to understand the function of TFRC in the survival of retinal cells that is relevant for glaucoma.

A small fraction of cellular TFRC is constitutively degraded in lysosomes and this

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process requires the function of Rab12, a GTPase involved in membrane vesicle trafficking. Rab12-mediated trafficking of TFRC from recycling endosomes to lysosomes is different than EGFR trafficking to lysosomes, which is mediated through classical endocytic pathways (Matsui et al., 2011). Our study showed that Rab12 is required for M98K-OPTN -induced

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cell death and autophagic degradation of TFRC. Rab12 also showed interaction with optineurin and colocalization with M98K-OPTN in autophagosomes. Moreover, knockdown of Rab12 inhibited autolysosome formation upon autophagy induction in retinal cells

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suggesting its role in autophagosome maturation to autolysosome (Sirohi et al., 2013).

M98K mutation enables a gain of function by interacting better with TFRC and causing its degradation in autophagosomes by enhanced recruitment of Rab12. However, how M98K-OPTN induces autophagy and its selectivity for death of retinal cells is still not

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known. These studies with M98K mutant were carried out using a cell culture model and it is essential to explore the role of M98K mutant in glaucoma pathogenesis using patient derived cells and transgenic animal models.

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10. Cytoprotective functions of optineurin:

A cytoprotective role of optineurin was speculated in the eye and optic nerve (Rezaie

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et al., 2002). Subsequently, it has been shown that depletion of optineurin results in reduced secretion of neurotrophin-3 (NT-3) and cilliary neurotrophic factor, which induces cell death in retinal cells. Exogenous supply of NT-3 inhibits retinal cell death (Sippl et al., 2011). Knockdown of optineurin results in activation of NF-κB, which induces cell death in neuronal cells as the inhibition of NF-κB signalling by its chemical inhibitor, withaferin, reduced this cell death (Akizuki et al., 2013). Optineurin translocates to nucleus upon apoptotic stimuli to prevent death of NIH3T3 cells whereas glaucoma-associated E50K mutant does not translocate to nucleus and make these cells susceptible to stress (De Marco et al., 2006). Cytoprotective function of optineurin has also been shown in as in vivo system.

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ACCEPTED MANUSCRIPT Depletion of optineurin in zebrafish embryos results in increased cell death, slight change in cell morphology, cell migration and defects in vesicle transport (Paulus and Link, 2014).

11. Autophagy and glaucoma Involvement of autophagy in glaucoma has been examined in animal models.

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Autophagy has protective effect on RGCs in mice subjected to optic nerve axotomy as shown by specific deletion of Atg5 in RGCs. Pharmacological induction of autophagy in this mouse model resulted in enhanced survival of RGCs, whereas specific deletion of Atg5 in RGCs reduced cell survival (Rodriguez-Muela et al., 2012). During ageing, macro-autophagy

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decreased in the retina whereas chaperon-mediated autophagy (CMA) increased. This increase in CMA was also seen in RGCs from Atg5 deletion mice, showing a cross talk between macro-autophagy and CMA (Rodriguez-Muela et al., 2013). Autophagy also

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protects retina from light-induced degeneration (Chen et al., 2013).

In contrast to the protective function of autophagy in RGCs in optic nerve axotomy model, Park et al., have suggested that autophagy mediates RGC death in glaucoma induced by chronic elevation of IOP (Park et al., 2012). Upon elevation of IOP, formation of autophagosomes and cell death was seen in RGCs. Treatment with an autophagy inhibitor

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reduced this cell death. In optic nerve crush model of acute axonal degeneration, autophagy was calcium dependent which mediated RGC death (Knoferle et al., 2010). Thus, depending upon the type of stress or insult, autophagy can either prevent or mediate RGC death in glaucoma models. Role of autophagy in glaucoma and other eye diseases has been discussed

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in an excellent review (Frost et al., 2014).

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12. Autophagy and apoptosis

It is obvious that autophagy plays an important role in the survival of retinal cells

because a decrease or increase in autophagy, as seen with E50K-OPTN and M98K-OPTN, respectively, causes apoptotic cell death (Chalasani et al., 2014; Sirohi et al., 2013). How autophagy is linked with apoptosis is not clear. Molecular components involved in autophagy are different from those involved in apoptosis. The cross-talk between autophagy and apoptosis is somewhat complex, and, depending upon cellular context and inducer, autophagy can induce or inhibit apoptotic cell death (Thorburn, 2014). Molecular mechanisms involved in autophagy-dependent apoptosis are yet to be explored in retinal cells. M98K-OPTNinduced autophagy leads to TFRC degradation subsequently resulting in apoptosis. Since overexpression of TFRC prevents apoptotic cell death, it is likely that TFRC has a role in

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ACCEPTED MANUSCRIPT anti-apoptotic signaling. Another possibility is that M98K-OPTN-induced autophagy mediates degradation of an anti-apoptotic protein and TFRC overexpression may be preventing this degradation. In case of E50K-OPTN-induced inhibition of autophagy, decrease in degradation of a pro-apoptotic protein would be expected to result in apoptosis. Thus, the nature of the protein molecule (pro-apoptotic or anti-apoptotic) being degraded by

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autophagy would decide the survival of the cell.

13. Future directions

A role for optineurin in autophagy in retinal cells is clearly established. Impaired

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autophagy is one of the mechanisms which possibly contribute to RGC death in glaucoma caused by optineurin mutants (Fig. 4). However, the molecular mechanisms involved in M98K-OPTN-mediated enhanced autophagy and E50K-OPTN-mediated block in autophagy

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are not clear. Several questions remain unanswered. How does M98K mutation enhance autophagy? Does phosphorylation at Ser177 play a role in this process? The function of ubiquitin-binding domain is required for M98K-OPTN and E50K-OPTN-induced autophagic cell death, but the role of ubiquitination, the ligases and their substrates involved in this process, are yet to be identified. TBK1, a glaucoma-associated protein involved in autophagy,

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directly interacts with optineurin but how TBK1 and OPTN interact functionally in retinal cells is yet to be explored. We also need to understand the molecular mechanisms that are involved in caspase activation and apoptosis when autophagy is altered by optineurin mutants. Finally, other survival mechanisms, such as secretion of neurotrophins, may get

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affected by loss of optineurin function. Is there any link between secretion of neurotrophins and autophagy? This needs to be explored because autophagy is involved in secretion of

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certain proteins. The data obtained with cell lines need to be verified using patient derived cells or transgenic animal models.

Acknowledgments

GS gratefully acknowledges the Department of Science and Technology, Government of India for J.C. Bose National Fellowship. Work in the GS laboratory has been supported by grants (BSC0208 and BSC0115) from CSIR, New Delhi, India. KS is thankful to CSIR, New Delhi, India for a research fellowship.

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Figure legends:

Figure 1: Disease associated mutations, functions and interactions of optineurin.

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Domain structure of human optineurin protein and mutations associated with glaucoma and ALS are shown. The figure also depicts the optineurin interacting proteins and other proteins

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involved in optineurin-mediated cellular functions.

Figure 2: Possible modes of action of E50K-OPTN leading to retinal cell death. Optineurin protein interacts with the Rab GTPase activating protein, TBC1D17. E50K mutation in optineurin alters its interaction with TBC1D17 and this in turn affects its downstream roles such as inhibition of Rab GTPases which leads to impaired transferrin receptor recycling and inhibition of autophagy, thus, causing loss of homeostasis and cell

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death. Altered interaction of E50K-OPTN with TBK1 results in E50K-OPTN insolubility and this might contribute to cell death. E50K-OPTN causes reduced proteasomal activity which is likely to result in loss of homeostasis. E50K-OPTN also generates ROS possibly by mitochondrial damage that leads to cell death.

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Figure 3: Model shows how M98K-OPTN alters cellular transferrin receptor dynamics leading to death of retinal cells. Under steady-state conditions, TFRC-transferrin-Fe

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complex from the plasma membrane is internalized by endocytosis and TFRC-transferrin is recycled back after intracellular release of Fe. Some of the TFRC molecules undergo lysosomal degradation through autophagy. In the presence of M98K-OPTN, recycling of TFRC is altered as M98K-OPTN interacts more strongly with TFRC, and enhances its localization in autophagosomes. M98K-OPTN recruits Rab12, a GTPase involved in TFRC degradation, more efficiently. As a consequence, cellular TFRC is rapidly degraded through autophagy, rather than being recycled to the plasma membrane through recycling endosomes. Figure 4: Overview of the possible mechanisms leading to disease condition by mutated optineurin. Mutation or altered levels of optineurin could lead to defects in retinal cells and/or glial cells (activation by optineurin deregulation) resulting in retinal cell death.

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Highlights

Mutations in OPTN cause glaucoma (E50K, M98K) and ALS.

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E50K-OPTN impairs autophagy, vesicle traffic and causes aggregate formation.

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E50K-OPTN-induced block in autophagy is dependent on a GTPase activating protein.

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M98K-OPTN induces Rab12-dependent autophagy that leads to retinal cell death.

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