Hongyu Ying

CELLULAR AND MOLECULAR BIOLOGY OF OPTINEURIN

Optineurin is a gene linked to glaucoma, amyotrophic lateral sclerosis, other neurodegenerative diseases, and Pagets disease of bone. This review describes the characteristics of optineurin and summarizes the cellular and molecular biology investigations conducted so far on optineurin. Data from a number of laboratories indicate that optineurin is a cytosolic protein containing 577 amino acid residues. Interacting with proteins such as myosin VI, Rab8, huntingtin, transferrin receptor, and TANK-binding kinase 1, optineurin is involved in basic cellular functions including protein trafficking, maintenance of the Golgi apparatus, as well as NF-κB pathway, antiviral, and antibacteria signaling. Mutation or alteration of homeostasis of optineurin (such as overexpression or knockdown) results in adverse consequences in the cells, leading to the development of neurodegenerative diseases including glaucoma.

Processing of Optineurin in Neuronal Cells

Optineurin is a gene linked to amyotrophic lateral sclerosis, Paget disease of bone, and glaucoma, a major blinding disease. Mutations such as E50K were identified in glaucoma patients. We investigated herein the involvement of ubiquitin-proteasome pathway (UPP) and autophagy, two major routes for protein clearance, in processing of optineurin in a retinal ganglion cell model line RGC5 and neuronal PC12 cells. It was found that the endogenous optineurin level in neuronal cells was increased by treatment of proteasomal inhibitor but not by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pulldown, indicating that optineurin was ubiquitinated. In cells overexpressing wild type and E50K optineurin, the level of the proteasome regulatory β5 subunit (PSMB5, indicative of proteasome activity) was reduced, whereas that for autophagy marker microtubule-associated protein 1 light chain 3 was enhanced compared with controls. Autophagosome formation was detected by electron microscopy. The foci formed after optineurin transfection were increased upon treatment of an autophagic inhibitor but were decreased by treatment of an inducer, rapamycin. Moreover, the level of optineurin-triggered apoptosis was reduced by rapamycin. This study thus provides compelling evidence that in a normal homeostatic situation, the turnover of endogenous optineurin involves mainly UPP. When optineurin is up-regulated or mutated, the UPP function is compromised, and autophagy comes into play. A decreased PSMB5 level and an induced autophagy were also demonstrated in vivo in retinal ganglion cells of E50K transgenic mice, validating and making relevant the in vitro findings.

Impairment of protein trafficking upon overexpression and mutation of optineurin.

Background Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu50 to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis. Methodology/Principal Findings Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu157 to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization. Conclusions/Significance The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.

Posttranslational modifications, localization, and protein interactions of optineurin, the product of a glaucoma gene

Background Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin. Methodology/Principal Findings Lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation or membrane protein extraction. The phosphorylation status was evaluated after immunoprecipitation. It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. RGC5 and human retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with molecules including Rab8, myosin VI, and transferrin receptor to assemble into supermolecular complexes. When overexpressed, optineurin–green fluorescence protein (GFP) fusion protein formed punctate structures termed “foci” in the perinuclear region. Treatment of nocadazole resulted in dispersion of the optineurin foci. In addition, tetracycline-regulated optineurin-GFPs expressing RGC5 stable cell lines were established for the first time. Conclusions/Significance The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology. The inducible optineurin-GFP–expressing cell lines are also anticipated to facilitate in-depth studies of optineurin. Furthermore, the demonstrations that optineurin is an aggregation-prone protein and that the foci formation is microtubule-dependent bear similarities to features documented in neurodegenerative diseases, supporting a neurodegenerative paradigm for glaucoma.

Optineurin: The autophagy connection

Optineurin is a cytosolic protein encoded by the OPTN gene. Mutations of OPTN are associated with normal tension glaucoma and amyotrophic lateral sclerosis. Autophagy is an intracellular degradation system that delivers cytoplasmic components to the lysosomes. It plays a wide variety of physiological and pathophysiological roles. The optineurin protein is a selective autophagy receptor (or adaptor), containing an ubiquitin binding domain with the ability to bind polyubiquitinated cargoes and bring them to autophagosomes via its microtubule-associated protein 1 light chain 3-interacting domain. It is involved in xenophagy, mitophagy, aggrephagy, and tumor suppression. Optineurin can also mediate the removal of protein aggregates through an ubiquitin-independent mechanism. This protein in addition can induce autophagy upon overexpression or mutation. When overexpressed or mutated, the optineurin protein also serves as a substrate for autophagic degradation. In the present review, the multiple connections of optineurin to autophagy are highlighted.

Angiogenesis in glaucoma filtration surgery and neovascular glaucoma: A review.

Angiogenesis may pose a clinical challenge in glaucoma, for example, during the wound healing phase after glaucoma filtration surgery and in the severe secondary glaucoma called neovascular glaucoma (NVG). Upregulation of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, occurs in eyes that have undergone glaucoma filtration surgery, as well as those with NVG. This has led investigation of the ability of anti-vascular endothelial growth factor therapy to improve outcomes, and we examine the findings with respect to the safety and efficacy of anti-vascular endothelial growth factor agents, mainly bevacizumab and ranibizumab, in eyes that have undergone glaucoma filtration surgery or have NVG. Combining conventional therapies-such as antimetabolites after filtration surgery and panretinal photocoagulation in NVG-and anti-vascular endothelial growth factor drugs may produce a synergetic effect, although further studies are required to evaluate the long-term efficacy of combination treatments.

Effects of mutations and deletions in the human optineurin gene

Optineurin is a gene associated with normal tension glaucoma (NTG) and amyotrophic lateral sclerosis (ALS). Foci formation and functional consequences including Golgi fragmentation, impairment of vesicle trafficking and apoptosis were observed previously upon overexpression and/or mutation of optineurin.In the current study, a total of 15 GFP tagged constructs that included NTG (E50K and 2 bp-AG insertion), ALS (exon 5 deletion, R96L, Q398X, and E478G) and non-disease (L157A and D474N) associated mutants and a series of deletion fragments were cloned into mammalian expression vectors and transfected into RGC5 and/or Neuro2A cells to evaluate whether their expression confer the optineurin phenotypes. The cells were monitored for foci formation and stained by immunofluorescence with anti-GM130 to analyze the Golgi integrity. Transferrin uptake experiments were performed to evaluate the protein trafficking process and apoptosis was assessed with the active caspase 3/7 detection kit. We demonstrated that cells expressing E50K and R96L optineurin exhibited all of the optineurin phenotypes. Q398X mutant did not induce foci formation, but triggered Golgi fragmentation, impairment of transferrin uptake and increase in apoptosis. The 2 bp-AG insertion mutant had a nuclear localization, compromised the transferrin uptake and strongly induced apoptosis. The foci formation, which might not predict the rest of the phenotypes, appeared to require both the leucine zipper and ubiquitin binding domains of the optineurin sequence. Interactions of optineurin with proteins including Rab8, myosin VI, huntingtin and transferrin receptor might directly determine whether the Golgi and protein trafficking phenotypes would be manifested. Examination of mutants and deletion fragments located at various sites of optineurin gene provide clues as to what regions of the gene may play a critical role in the development of pathologic consequences.

Wnt Activation by Wild Type and Mutant Myocilin in Cultured Human Trabecular Meshwork Cells

Background Myocilin is a gene linked to the most prevalent form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The Pro370 to Leu (P370L) mutation of myocilin is associated with severe glaucoma phenotypes and Gln368 stop (Q368X) is the most common myocilin mutation reported. Myocilin, upon overexpression, has been shown to induce phenotypes that include a loss of actin stress fibers, an increase in the cAMP level and protein kinase A (PKA) activity, as well as a reduction in the RhoA activity. We examined herein whether Wnt signaling pathway is involved in the myocilin phenotypes and whether P370L and Q368X mutants also display biological effects similar to those of the wild type myocilin. Methodology/Principal Findings Wild type myocilin, when transfected into cultured human TM cells, induced a loss of actin stress fibers as judged by phalloidin staining. Such a loss was averted by treatment of secreted Frizzled-related protein 1 (sFRP1), an inhibitor of Wnt signaling. Consistent with the notion that Wnt pathway mediates the myocilin phenotype, Wnt activation was demonstrated by TOP/FOP-Flash reporter assays. Treatment of human TM cells of a Wnt activator, SB216763, as well as transfection of myocilin P370L and Q368X mutants all resulted in actin stress fiber loss, PKA activation and RhoA inactivation. The PKA elevation was obviated by the sFRP1 treatment, indicating that Wnt signaling was upstream that of PKA. Conclusions/Significance The present study demonstrated that following forced expression of wild type myocilin, Wnt was activated, triggering in turn other myocilin-related alterations. P370L and Q368X mutations induced similar phenotypes, suggesting one possible mechanism how the mutants may lead to TM cell damage and pathology.

Cellular Processing of Myocilin

Background Myocilin (MYOC) is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Pro370Leu (P370L) and Gln368stop (Q368X) have been identified in patients. In the present study, we investigated the processing of myocilin in human trabecular meshwork (TM) cells as well as in inducible, stable RGC5 cell lines. Methodology/Principal Findings The turnover and photoactivation experiments revealed that the endogenous myocilin in human trabecular meshwork (TM) cells was a short-lived protein. It was found that the endogenous myocilin level in TM cells was increased by treatment of lysosomal and proteasomal inhibitors, but not by autophagic inhibitor. Multiple bands immunoreactive to anti-ubiquitin were seen in the myocilin pull down, indicating that myocilin was ubiquitinated. In inducible cell lines, the turnover rate of overexpressed wild-type and mutant P370L and Q368X myocilin-GFP fusion proteins was much prolonged. The proteasome function was compromised and autophagy was induced. A decreased PSMB5 level and an increased level of autophagic marker, LC3, were demonstrated. Conclusions/Significance The current study provided evidence that in normal homeostatic situation, the turnover of endogenous myocilin involves ubiquitin-proteasome and lysosomal pathways. When myocilin was upregulated or mutated, the ubiquitin-proteasome function is compromised and autophagy is induced. Knowledge of the degradation pathways acting on myocilin can help in design of novel therapeutic strategies for myocilin-related glaucoma.

The Role of crumbs Genes in the Vertebrate Cornea

PURPOSE To evaluate the role of crumbs genes and related epithelial polarity loci in the vertebrate cornea. METHODS The authors used histologic analysis and electron microscopy to evaluate the corneas of zebrafish mutant for a crumbs locus oko meduzy (ome) and in mutants of four other loci, nagie oko (nok), heart and soul (has), mosaic eyes (moe), and ncad (formerly glass onion), that function in the same or related genetic pathways. In parallel, they performed an evaluation of corneas in human carriers of a crumbs gene, CRB1, and mutations using topography and biomicroscopy. The expression of the CRB1 gene in the normal human cornea was examined by polymerase chain reaction (PCR) and immunohistochemical staining. RESULTS The corneas of zebrafish mutants display severe abnormalities of the epithelial and stromal layers. The epithelial cells do not properly adhere to each other, and fluid-filled spaces form between them. In addition, the layering of the corneal stroma is poorly formed or absent. The corneas of human carriers of CRB1 mutations display shape deviations compared with what has been observed in normal individuals. A PCR product of the correct size was obtained from normal human corneal samples. Sequence analyses confirmed its identity to be the human CRB1 gene. Immunohistochemical staining using anti-CRB1 yielded positive brown deposits in the human cornea. CONCLUSIONS crumbs genes play a role in the differentiation of the vertebrate cornea. Corneal defects associated with crumbs gene mutations are very severe in the zebrafish model and, in comparison, appear clinically less pronounced in the human eye.