Optineurin Expression Research Articles

Optineurin-mediated mitophagy protects renal tubular epithelial cells against accelerated senescence in diabetic nephropathy

Premature senescence is a key process in the progression of diabetic nephropathy (DN). Premature senescence of renal tubular epithelial cells (RTEC) in DN may result from the accumulation of damaged mitochondria. Mitophagy is the principal process that eliminates damaged mitochondria through PTEN-induced putative kinase 1 (PINK1)-mediated recruitment of optineurin (OPTN) to mitochondria. We aimed to examine the involvement of OPTN in mitophagy regulation of cellular senescence in RTEC in the context of DN. In vitro, the expression of senescence markers P16, P21, DcR2, SA-β-gal, SAHF, and insufficient mitophagic degradation marker (mitochondrial P62) in mouse RTECs increased after culture in 30 mM high-glucose (HG) conditions for 48 h. Mitochondrial fission/mitophagy inhibitor Mdivi-1 significantly enhanced RTEC senescence under HG conditions, whereas autophagy/mitophagy agonist Torin1 inhibited cell senescence. MitoTempo inhibited HG-induced mitochondrial reactive oxygen species and cell senescence with or without Mdivi-1. The expression of PINK1 and OPTN, two regulatory factors for mitophagosome formation, decreased significantly after HG stimulation. Overexpression of PINK1 did not enhance mitophagosome formation under HG conditions. OPTN silencing significantly inhibited HG-induced mitophagosome formation, and overexpression of OPTN relieved cellular senescence through promoting mitophagy. In clinical specimens, renal OPTN expression was gradually decreased with increased tubulointerstitial injury scores. OPTN-positive renal tubular cells did not express senescence marker P16. OPTN expression also negatively correlated with serum creatinine levels, and positively correlated with eGFR. Thus, OPTN-mediated mitophagy plays a crucial regulatory role in HG-induced RTEC senescence in DN. OPTN may, therefore, be a potential antisenescence factor in DN.

Overexpression of parkin protects retinal ganglion cells in experimental glaucoma

Glaucoma is a leading cause of irreversible blindness and characterized by progressive damage of retinal ganglion cells (RGCs). Growing evidences have linked impaired mitophagy with neurodegenerative diseases, while the E3 ubiquitin ligase parkin may play a key role. However, the pathophysiological relationship between parkin and glaucoma remains largely unknown. Using chronic hypertensive glaucoma rats induced by translimbal laser photocoagulation, we show here that the protein level of parkin and its downstream optineurin proteins were increased in hypertensive retinas. The ratio of LC3-II to LC3-I, the number of mitophagosomes, and unhealthy mitochondria were increased in hypertensive optic nerves. Overexpression of parkin by viral vectors increased RGC survival in glaucomatous rats in vivo and under excitotoxicity in vitro. It also promoted optineurin expression and improved mitochondrial health. In parkin-overexpressed glaucomatous rats, the ratio of LC3-II to LC3-I, LAMP1 level, and the number of mitophagosomes in optic nerve were decreased at 3 days, yet increased at 2 weeks following intraocular pressure (IOP) elevation. These findings demonstrate that dysfunction of mitophagy exist in RGCs of glaucomatous rats. Overexpression of parkin exerted a significant protective effect on RGCs and partially restored dysfunction of mitophagy in response to cumulative IOP elevation.

Co-expression network analysis of lncRNAs and mRNAs in OPTN-silenced cells.

The aim of the present study was to reveal the role of long noncoding RNAs(lncRNAs) in the regulation of the pathogenesis of optineurin(OPTN)-silenced cells. The microarray data set GSE12452 was re-annotated using the non-coding RNA function annotation server to identify differentially expressed lncRNAs. Weighted correlation network analysis was used to construct an lncRNA-lncRNA co-expression network and identify co-expression modules. Three OPTN small interfering RNAs (siRNAs) were transiently transfected into HeLa cells. Reverse transcription‑quantitative polymerase chain reaction(RT-qPCR) and western blot analysis were used to detect OPTN expression and select the most effective OPTNsiRNA to construct stably transfected cells. RT-qPCR was used to quantify the identified lncRNAs in the OPTN-silenced cells. The potential functions of these modules were explored by the functional enrichment of the corresponding co-expressed genes. A total of 3,495lncRNAs were re-annotated. Of these, matrix metalloprotease12 andRP11-169D4.1 were upregulated, and RP1-212P9.2 was downregulated. The results of the RT-qPCR analysis of RP1-212P9.2 andRP11‑169D4.1 were consistent with the re-annotated data in the OPTN‑silenced cells. GeneOntology analyses indicated that the biological functions of the mRNAs co-expressed with these lncRNAs were associated with gene product regulation, and neuronal migration, polarity and differentiation. In addition, Kyoto Encyclopedia of Genes and Genomes analysis indicated that the two validated lncRNAs were associated with the transforming growth factor-β signaling pathway and the apoptosis pathway, respectively. In conclusion, the abnormal lncRNAs identified in OPTN-silenced cells indicate that lncRNAs may contribute to the molecular pathogenesis of OPTN-associated diseases.

Expression of optineurin isolated from rat-injured dental pulp and the effects on inflammatory signals in normal rat kidney cells.

We previously isolated rat 14.7K-interacting protein-2 (rFIP-2) from the rat-wounded pulp. The protein, homologous to human FIP-2, is known as optineurin and was initially identified as a novel tumor necrosis factor-α (TNF-α)-inducible protein, and more recently, as an autophagy receptor. However, the biological role of optineurin in dental pulp remains elusive. We hypothesized that optineurin has a crucial role in regulating molecular processes during pulp inflammatory responses induced by TNF-α. We examined the kinetics of optineurin expression in pulp inflammation. Optineurin localization and expression were examined using rat pulp fibroblasts. The cells were treated with pharmacological inhibitors for TNF-α-induced inflammatory signals or with hydrogen peroxide as apoptotic stimuli. Stable optineurin-knockdown cells (OPTN-KD cells) were established by transfecting normal rat kidney cells with a vector expressing optineurin-specific small interfering RNA. Cell proliferation and the profiles of cytokines and intracellular signaling molecules were examined using OPTN-KD cells stimulated by TNF-α. Optineurin was localized in the cytoplasm and then translocated into the nucleus upon apoptotic stimuli. Optineurin expression was increased by TNF-α and decreased by a specific inhibitor of c-Jun N-terminal kinase. The OPTN-KD cells secreted smaller amounts of monocyte chemotactic protein-1 (MCP-1) and intracellular MCP-1 mRNA, and cell proliferation was significantly increased. Apoptosis-related signaling molecules were downregulated in OPTN-KD cells. These results demonstrated that optineurin is a crucial molecule mediated by TNF-α, which induces the production of inflammatory factors and apoptosis signaling, suggesting the presence of signaling interactions between optineurin and a transcription factor for MCP-1.

Decreased expression of miR-9 due to E50K OPTN mutation causes disruption of the expression of BDNF leading to RGC-5 cell apoptosis

The aims of the present study were to investigate the effect of E50K optineurin (OPTN) mutation on RGC‑5 cells and to define the role of microRNA‑9 (miR‑9) in this system. Transfected RGC‑5 cells were used to evaluate the effects of E50K OPTN on the expression of miR‑9 and subsequent disruption of RGC‑5 cell apoptosis was analyzed using western blotting. The results showed that the expression of E50K OPTN was associated with a marked reduction in the levels of miR‑9 in the E50K OPTN‑transfected RGC‑5 cells. The E50K OPTN‑dependent reductions in miR‑9 led to increased expression of the transcriptional repressor, RE1‑silencing transcription factor and decreased the expression of brain‑derived neurotrophic factor. Thus, E50K OPTN may disrupt the expression of miR‑9, suggesting a potential mechanism by which E50K OPTN mutation may lead to RGC‑5 cell apoptosis.

Optineurin expression in the nigrostriatal dopamine system: Normal expression and response to rotenone treatment

Mutations in the optineurin gene (OPTN) have been reported in rare familial and sporadic amyotrophic lateral sclerosis (ALS) cases. It is yet to be established whether mutations segregate with dominantly inherited familial ALS. We therefore performed mutation analysis in a cohort of 96 autosomal dominant ALS families. A novel heterozygous nonsynonymous variant (c.218C > T, S73L) was identified in one patient; however, analysis in the extended pedigree demonstrated that this variant was inherited from an unaffected parent. The variant was absent in 480 control individuals. The affected serine residue is highly conserved and its substitution is predicted to alter phosphorylation. Despite this, our evidence indicates that this variant is unlikely to play a pathogenic role in the disease. Cell and animal models will be required to functionally support the pathogenic role of OPTN mutations.

From the Cover: Alterations in Optineurin Expression and Localization in Pre-clinical Parkinson's Disease Models.

Parkinson's disease (PD) is a progressive neurodegenerative disease that affects ∼5 million people around the world. PD etiopathogenesis is poorly understood and curative or disease modifying treatments are not available. Mechanistic studies have identified numerous pathogenic pathways that overlap with many other neurodegenerative diseases. Mutations in the protein optineurin (OPTN) have recently been identified as causative factors for glaucoma and amyotrophic lateral sclerosis. OPTN has multiple recognized roles in neurons, notably in mediating autophagic flux, which has been found to be disrupted in most neurodegenerative diseases. OPTN(+ )aggregates have preliminarily been identified in cytoplasmic inclusions in numerous neurodegenerative diseases, however, whether OPTN has a role in PD pathogenesis has yet to be tested. Thus, we chose to test the hypothesis that OPTN expression and localization would be modulated in pre-clinical PD models. To test our hypothesis, we characterized midbrain OPTN expression in normal rats and in a rat rotenone PD model. For the first time, we show that OPTN is enriched in dopamine neurons in the midbrain, and its expression is modulated by rotenone treatment in vivo Here, animals were sampled at time-points both prior to overt neurodegeneration and after severe behavioral deficits, where a lesion to the nigrostriatal dopamine system is present. The effect and magnitude of OPTN expression changes are dependent on duration of treatment. Furthermore, OPTN colocalizes with LC3 (autophagic vesicle marker) and alpha-synuclein positive puncta in rotenone-treated animals, potentially indicating an important role in autophagy and PD pathogenesis.

Optineurin suppression activates the mediators involved in the terminal effector pathways of human labour and delivery

Spontaneous preterm birth remains the major cause of neonatal death and morbidity. Studies in non-gestational tissues report that optineurin (OPTN) is critical in the termination of NFKB1 activity and control of inflammation, central features of spontaneous preterm birth. The aims of the present study were to determine: (1) OPTN expression in fetal membranes and the myometrium during labour; (2) the effects of IL1B on OPTN expression in primary myometrial cells; and (3) the effects of OPTN short interference (si) RNA on IL1B-stimulated proinflammatory and prolabour mediators. OPTN mRNA and protein expression was significantly decreased with spontaneous term labour in fetal membranes and the myometrium. Although there was no effect of spontaneous preterm labour on OPTN expression in fetal membranes, there was decreased OPTN expression in membranes with chorioamnionitis and myometrial cells treated with 1ng mL-1 IL1B for 1 or 6h. In cells transfected with OPTN siRNA, significant increases were seen in IL1B-stimulated IL6, tumour necrosis factor, CXCL8 and monocyte chemoattractant protein-1 mRNA expression and release, cyclo-oxygenase-2 and prostanoid PTGFR receptor mRNA expression and the release of prostaglandin F2α. There was no change in IL1B-stimulated NFKBIA expression; however, there was increased NFKB1 p65 DNA-binding activity. The results of the present study suggest that OPTN is a negative regulator of inflammation-induced prolabour mediators.

Optineurin Negatively Regulates Osteoclast Differentiation by Modulating NF-κB and Interferon Signaling: Implications for Paget's Disease.

SummaryPaget’s disease of bone (PDB) is a common disease characterized by osteoclast activation that leads to various skeletal complications. Susceptibility to PDB is mediated by a common variant at the optineurin (OPTN) locus, which is associated with reduced levels of mRNA. However, it is unclear how this leads to the development of PDB. Here, we show that OPTN acts as a negative regulator of osteoclast differentiation in vitro and that mice with a loss-of-function mutation in Optn have increased osteoclast activity and bone turnover. Osteoclasts derived from Optn mutant mice have an increase in NF-κB activation and a reduction in interferon beta expression in response to RANKL when compared to wild-type mice. These studies identify OPTN as a regulator of bone resorption and are consistent with a model whereby genetically determined reductions in OPTN expression predispose to PDB by enhancing osteoclast differentiation.

Whole-genome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease.

Frontotemporal lobar degeneration with TAR DNA-binding protein 43 inclusions (FTLD-TDP) is the most common pathology associated with frontotemporal dementia (FTD). Repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) and mutations in progranulin (GRN) are the major known genetic causes of FTLD-TDP; however, the genetic etiology in the majority of FTLD-TDP remains unexplained. In this study, we performed whole-genome sequencing in 104 pathologically confirmed FTLD-TDP patients from the Mayo Clinic brain bank negative for C9ORF72 and GRN mutations and report on the contribution of rare single nucleotide and copy number variants in 21 known neurodegenerative disease genes. Interestingly, we identified 5 patients (4.8%) with variants in optineurin (OPTN) and TANK-binding kinase 1 (TBK1) that are predicted to be highly pathogenic, including two double mutants. Case A was a compound heterozygote for mutations in OPTN, carrying the p.Q235* nonsense and p.A481V missense mutation in trans, while case B carried a deletion of OPTN exons 13-15 (p.Gly538Glufs*27) and a loss-of-function mutation (p.Arg117*) in TBK1. Cases C-E carried heterozygous missense mutations in TBK1, including the p.Glu696Lys mutation which was previously reported in two amyotrophic lateral sclerosis (ALS) patients and is located in the OPTN binding domain. Quantitative mRNA expression and protein analysis in cerebellar tissue showed a striking reduction of OPTN and/or TBK1 expression in 4 out of 5 patients supporting pathogenicity in these specific patients and suggesting a loss-of-function disease mechanism. Importantly, neuropathologic examination showed FTLD-TDP type A in the absence of motor neuron disease in 3 pathogenic mutation carriers. In conclusion, we highlight TBK1 as an important cause of pure FTLD-TDP, identify the first OPTN mutations in FTLD-TDP, and suggest a potential oligogenic basis for at least a subset of FTLD-TDP patients. Our data further add to the growing body of evidence linking ALS and FTD and suggest a key role for the OPTN/TBK1 pathway in these diseases.

Rapamycin and Chloroquine: The In Vitro and In Vivo Effects of Autophagy-Modifying Drugs Show Promising Results in Valosin Containing Protein Multisystem Proteinopathy

Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB), frontotemporal dementia (FTD), more recently termed multisystem proteinopathy (MSP). Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43)-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs.

Effects of abnormal optineurin expression on the survival of the rat retinal ganglion cell line RGC-5.

The OPTN gene is thought to be associated with certain types of glaucoma and the function of the protein for which it encodes, optineurin, has been extensively researched, but with contradictory results. We explored the effects of abnormal optineurin expression on the survival of the rat retinal ganglion cell line RGC-5. Plasmids expressing wild-type (WT) or E50K mutant optineurin, or OPTN-specific double-hairpin small interfering RNA (si-RNA), were transfected into RGC-5 cells. The effects on cell survival were monitored by observation of cell morphology and propidium iodide and Hoechst 33342 fluorescent staining, while expression of optineurin was visualized by fluorescence microscopy. Abnormal optineurin expression influenced the survival of RGCs in vitro, as apoptosis was induced by increased WT and E50K mutant optineurin, while a reduction in apoptosis was observed in cells transfected with OPTN-siRNA. Similar results were also observed in transfected cells treated with apoptotic stimuli. Overexpression of WT and mutant E50K protein resulted in greater cell death, while downregulation decreased RGC-5 apoptosis.

Distribution and localization of abnormally expressed OPTN proteins in RGC5 retinal ganglion cells and their effects on subcellular morphology.

The objectives of this study were to investigate the distributions of abnormally expressed optineurin (OPTN) proteins in retinal ganglion cells (RGC5s) of transgenic rats and their effects on subcellular morphological structures. Green fluorescent protein labeled EGFP wild-type (OPTN(WT)), E50K mutant type (OPTN(E50K)), and OPTN siRNA (si-OPTN) eukaryotic expression plasmids were constructed and transfected into RGC5s. Intracellular structures were labeled with organelle specific fluorescent dyes. Construct localization and cell morphologies were visualized by confocal fluorescence microscopy. OPTN(WT) was observed to be distributed as fine punctate fluorescent particles in the cytoplasm around the nucleus, along with exhibiting nuclear expression. OPTNE50K exhibited similar distribution but with non-uniform fluorescence particle size. si-OPTN distribution was similar to that of EGFP: uniform across the cytoplasm and nucleus. Compared with the negative control group, OPTN(WT), and OPTN(E50K) and to a lesser degree pEGFP-transfected cells exhibited fracture and loss of myofilament proteins and mitochondrial swelling and cytoplasmic accumulation, along with abnormal lysosomal distribution and increased volume, and Golgi fragmentation. However, si- OPTN transfected cells exhibited no significant damage. Therefore, we demonstrated that the E50K mutation disrupts the uniformity of OPTN protein distribution upon exogenous overexpression. Furthermore, these results suggested that si-OPTN transfection, and thus potentially OPTN knockdown, did not impact subcellular morphology of RGC5 cells, whereas transfection, especially when combined with wild-type or mutant OPTN expression, led to substantial abnormalities in subcellular morphological structures. These findings lay a foundation for further research into the function of the OPTN protein.

Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis.

ABSTRACTCrohn's disease (CD) is associated with delayed neutrophil recruitment and bacterial clearance at sites of acute inflammation as a result of impaired secretion of proinflammatory cytokines by macrophages. To investigate the impaired cytokine secretion and confirm our previous findings, we performed transcriptomic analysis in macrophages and identified a subgroup of individuals with CD who had low expression of the autophagy receptor optineurin (OPTN). We then clarified the role of OPTN deficiency in: macrophage cytokine secretion; mouse models of bacteria-driven colitis and peritonitis; and zebrafish Salmonella infection. OPTN-deficient bone-marrow-derived macrophages (BMDMs) stimulated with heat-killed Escherichia coli secreted less proinflammatory TNFα and IL6 cytokines despite similar gene transcription, which normalised with lysosomal and autophagy inhibitors, suggesting that TNFα is mis-trafficked to lysosomes via bafilomycin-A-dependent pathways in the absence of OPTN. OPTN-deficient mice were more susceptible to Citrobacter colitis and E. coli peritonitis, and showed reduced levels of proinflammatory TNFα in serum, diminished neutrophil recruitment to sites of acute inflammation and greater mortality, compared with wild-type mice. Optn-knockdown zebrafish infected with Salmonella also had higher mortality. OPTN plays a role in acute inflammation and neutrophil recruitment, potentially via defective macrophage proinflammatory cytokine secretion, which suggests that diminished OPTN expression in humans might increase the risk of developing CD.

LB-003 The Role Of Optineurin In Macrophage Cytokine Secretion And Bowel Inflammation

published in/presented at:Presented at Keystone Symposia February 2014.IntroductionCrohn’s disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. We previously showed that macrophages from CD patients secrete lower levels...

IRF-1-binding site in the first intron mediates interferon-γ-induced optineurin promoter activation

Optineurin is an adaptor protein involved in signal transduction, membrane vesicle trafficking and autophagy. Optineurin expression is induced by cytokines. Previously we have shown that tumor necrosis factor-α activates optineurin promoter through NF-κB-binding site in the core promoter. However, this promoter was not activated by interferon-γ. Here, we report identification of a functional IRF-1-binding site in the first intron of human optineurin gene that mediates interferon-γ-induced activation of the promoter. Optineurin promoter, containing the contiguous intronic sequences with IRF-1 responsive sites, is strongly activated by IRF-1. Mutational inactivation of IRF-1 site resulted in loss of activation of the promoter by interferon-γ and also by IRF-1. We also show that IRF-1 cooperates with NF-κB to activate optineurin promoter. The synergistic effect of these two transcription factors (IRF-1 and NF-κB) may be involved in cooperative induction of optineurin promoter by interferon-γ and tumor necrosis factor-α.

Optineurin suppression causes neuronal cell death via NF‐κB pathway

Mutations in more than 10 genes are reported to cause familial amyotrophic lateral sclerosis (ALS). Among these genes, optineurin (OPTN) is virtually the only gene that is considered to cause classical ALS by a loss-of-function mutation. Wild-type optineurin (OPTN(WT) ) suppresses nuclear factor-kappa B (NF-κB) activity, but the ALS-causing mutant OPTN is unable to suppress NF-κB activity. Therefore, we knocked down OPTN in neuronal cells and examined the resulting NF-κB activity and phenotype. First, we confirmed the loss of the endogenous OPTN expression after siRNA treatment and found that NF-κB activity was increased in OPTN-knockdown cells. Next, we found that OPTN knockdown caused neuronal cell death. Then, overexpression of OPTN(WT) or OPTN(E) (50K) with intact NF-κB-suppressive activity, but not overexpression of ALS-related OPTN mutants, suppressed the neuronal death induced by OPTN knockdown. This neuronal cell death was inhibited by withaferin A, which selectively inhibits NF-κB activation. Lastly, involvement of the mitochondrial proapoptotic pathway was suggested for neuronal death induced by OPTN knockdown. Taken together, these results indicate that inappropriate NF-κB activation is the pathogenic mechanism underlying OPTN mutation-related ALS. Among the genes for typical amyotrophic lateral sclerosis (ALS) phenotypes, optineurin (OPTN) is virtually the only gene in which a loss-of-function mutation is considered as the principal disease mechanism. We found that OPTN knockdown induced neuronal cell death via NF-κB activation. Furthermore, proapoptotic molecules such as p53 and Bax representing downstream targets of NF-κB are suggested to be involved in neuronal death.

Optineurin in neurodegenerative diseases

Optineurin is a gene associated with normal tension glaucoma and primary open-angle glaucoma, one of the major causes of irreversible bilateral blindness. Recently, mutations in the gene encoding optineurin were found in patients with amyotrophic lateral sclerosis (ALS). Immunohistochemical analysis showed aggregation of optineurin in skein-like inclusions and round hyaline inclusions in the spinal cord, suggesting that optineurin appears to be a more general marker for ALS. However, our detailed examinations demonstrated that optineurin was found not only in ALS-associated pathological structures, but also in ubiquitin-positive intraneuronal inclusions in ALS with dementia, basophilic inclusions in the basophilic type of ALS, neurofibrillary tangles and dystrophic neurites in Alzheimer's disease, Lewy bodies and Lewy neurites in Parkinson's disease, ballooned neurons in Creutzfeldt-Jakob disease, glial cytoplasmic inclusions in multiple system atrophy, and Pick bodies in Pick disease. With respect to optineurin-positive basophilic inclusions, these structures showed variable immunoreactivities for ubiquitin; some structures were obviously ubiquitin-positive, while others were negative for the protein, suggesting that optineurin expression was not always associated with the expression of ubiquitin. This study indicates that optineurin is widely distributed in neurodegenerative conditions; however, its significance is obscure.

NF-κB Mediates Tumor Necrosis Factor α-Induced Expression of Optineurin, a Negative Regulator of NF-κB

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor κB (NF-κB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor α (TNFα). Mutation of a putative NF-κB-binding site present in the core promoter resulted in loss of basal as well as TNFα-induced activity. Overexpression of p65 subunit of NF-κB activated this promoter through NF-κB site. Oligonucleotides corresponding to this putative NF-κB-binding site showed binding to NF-κB. TNFα-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-κB (IκBα) super-repressor. Blocking of NF-κB activation resulted in inhibition of TNFα-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFα-induced NF-κB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFα-induced as well as basal NF-κB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-κB pathway in response to TNFα. In addition these results suggest that there is a negative feedback loop in which TNFα-induced NF-κB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-κB.

Functional analysis of mutants of the optineurin gene, associated with some forms of glaucoma

Abstract Purpose Mutations in the gene OPTN are associated with normal tension and open angle glaucomas. We have studied the effects of some of these mutations on the cellular biology of retinal ganglion cells, and tried to infer the role of the protein optineurin. Methods We transfected plasmids expressing normal or wild‐type (WT) and E50K, R545Q, H26D, and H486R mutant optineurin into a variety of cells such as HeLa, COS‐1, retinal pigment epithelial (RPE), and the rat retinal ganglion cell (RGC) line RGC‐5, and followed their effects on cell survival by morphologic observation of cells. Expression of optineurin and its mutants was monitored by immunofluorescence staining of cells and by Western blotting. Results The E50K mutant of optineurin, which is associated with the severest phenotype, was seen to selectively induce the death of retinal ganglion cells but not of the other cell lines tested. Neither the wild type cDNA nor the other mutants have any such effect. This cell death induced by E50K OPTN was inhibited by the antioxidants N‐acetylcysteine and Trolox. E50K was seen to generate reactive oxygen species (ROS), which were reduced by antioxidants. Coexpression of manganese superoxide dismutase with the E50K mutant abolished ROS production and inhibited cell death. Conclusion E50K optineurin is a gain of function mutant, which has acquired the ability to induce cell death selectively in retinal ganglion cells. This cell death was mediated by oxidative stress. The present findings suggest the possibility of antioxidant use for delaying or controlling some forms of glaucoma.