Characterization of a Novel Mouse Model for Fuchs Endothelial Corneal Dystrophy.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is a progressive blinding disorder, characterized by increased corneal endothelial excrescences (guttae), corneal endothelial cell loss, and edema. These symptoms are hypothesized to be caused by changes in the extracellular matrix (ECM) and mitochondrial dysfunction in the corneal endothelium. Despite this clinical and biological relevance, a comprehensive animal model that recapitulates all the major disease characteristics is currently unavailable. In this study, we develop such a model to improve our understanding of the signaling pathways involved in the FECD progression and develop strategies for early intervention. Method: To generate a comprehensive FECD model, we generated a double mutant mouse bearing tamoxifen-inducible knockdown of Slc4a11 and the Col8a2 (Q455K) mutation. We performed optical coherence tomography (OCT) and in vivo confocal microscopy using the Heidelberg Retinal Tomography 3 - Rostock Cornea module (HRT3-RCM) on the mice at 5 weeks of age before tamoxifen feeding to establish baseline values for corneal thickness, endothelial cell density, and test for the presence of guttae. We measured these parameters again post-tamoxifen treatment at 16 weeks of age. We collected corneas at 16 weeks to perform histopathology, immunofluorescence staining for tight junctions, adherens junctions, and oxidative stress. We evaluated endothelial pump function using a lactate assay. Results: The double mutant tamoxifen-fed animals showed the presence of guttae, and displayed increased corneal thickness and decreased endothelial cell density. Endothelial cells showed altered morphology with disrupted adherens junctions and elevated reactive oxygen species (ROS). Finally, we found that stromal lactate concentrations were elevated in the double mutant mice, indicative of compromised endothelial pump function. Conclusions: Overall, this mouse model recapitulates all the important phenotypic features associated with FECD.

AUTOPHAGY IN THE EYE: FROM PHYSIOLOGY TO PATHOPHYSOLOGY.

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.

Rescue of the Congenital Hereditary Endothelial Dystrophy Mouse Model by Adeno-Associated Viruse-Mediated Slc4a11 Replacement.

Purpose: Congenital hereditary endothelial dystrophy (CHED) is a rare condition that manifests at an early age showing corneal edema, increased oxidative stress, mitochondrial dysfunction, and eventually apoptosis of the endothelium due to loss of function of the membrane transport protein SLC4A11. This project tested whether replacing Slc4a11 into the Slc4a11 -/- CHED mouse model can reverse the disease-associated phenotypes. Design: Experimental study. Participants: Five-week-old or 11-week-old Slc4a11 -/- mice. Age- and gender-matched Slc4a11 +/+ animals were used as controls. A total of 124 animals (62 female, and 62 male) were used in this study. Fifty-three animals of the genotype Slc4a11 +/+ were used as age- and gender-matched noninjected controls. Seventy-one Slc4a11 -/- mice were administered anterior chamber injections of adeno-associated virus (AAV). Methods: Anterior chambers of young (5 weeks old) or older (11 weeks old) Slc4a11 -/- mice were injected once with adeno-associated virus serotype 9 (AAV9) mouse Slc4a11 or AAV9-Null vectors. Corneal thickness was measured using OCT. End point analysis included corneal endothelial cell density, mitochondrial oxidative stress, and corneal lactate concentration. Main Outcome Measures: Corneal thickness, endothelial cell loss, lactate levels, and mitochondrial oxidative stress. Results: In the young animals, AAV9-Slc4a11 reversed corneal edema, endothelial cell loss, mitochondrial oxidative stress, lactate transporter expression, and corneal lactate concentration to the levels observed in wild-type animals. In the older animals, gene replacement did not reverse the phenotype but prevented progression. Conclusions: Functional rescue of CHED phenotypes in the Slc4a11 -/- mouse is possible; however, early intervention is critical.

Mitochondrial ROS in Slc4a11 KO Corneal Endothelial Cells Lead to ER Stress.

Recent studies from Slc4a11 -/- mice have identified glutamine-induced mitochondrial dysfunction as a significant contributor toward oxidative stress, impaired lysosomal function, aberrant autophagy, and cell death in this Congenital Hereditary Endothelial Dystrophy (CHED) model. Because lysosomes are derived from endoplasmic reticulum (ER)-Golgi, we asked whether ER function is affected by mitochondrial ROS in Slc4a11 KO corneal endothelial cells. In mouse Slc4a11 -/- corneal endothelial tissue, we observed the presence of dilated ER and elevated expression of ER stress markers BIP and CHOP. Slc4a11 KO mouse corneal endothelial cells incubated with glutamine showed increased aggresome formation, BIP and GADD153, as well as reduced ER Ca2+ release as compared to WT. Induction of mitoROS by ETC inhibition also led to ER stress in WT cells. Treatment with the mitochondrial ROS quencher MitoQ, restored ER Ca2+ release and relieved ER stress markers in Slc4a11 KO cells in vitro. Systemic MitoQ also reduced BIP expression in Slc4a11 KO endothelium. We conclude that mitochondrial ROS can induce ER stress in corneal endothelial cells.

Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy.

Purpose: The Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice. Methods: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ. Results: Even though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss. Conclusions: Mitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.

Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.

Purpose: The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity. Methods: Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks. Corneal thickness (CT) was measured by OCT. At eight weeks endpoint, oxidative damage, tight junction integrity, stromal lactate concentration, endothelial permeability, differentially expressed transporters, and junction proteins were determined. Separately, a keratocyte only inducible Slc4a11 KO was also examined. Results: At four weeks post-Tm induction Slc4a11 transcript levels were 2% of control. Corneal thickness increased gradually and was 50% greater than Wild Type (WT) after eight weeks with significantly altered endothelial morphology, increased nitrotyrosine staining, significantly higher stromal lactate, decreased expression of lactate transporters and Na-K ATPase activity, higher ATP, altered expression of tight and adherens junctions, and increased fluorescein permeability. No significant differences in CT were found between WT and keratocyte only Slc4a11 KO. Conclusions: The Slc4a11 inducible KO shows development of a similar phenotype as the conventional KO, thereby validating the model and providing a tool for further use in examining the sequence of cellular events by use of noninvasive in vivo physiological probes.

Bicarbonate activates glycolysis and lactate production in corneal endothelial cells by increased pHi.

Recent studies have shown that lactate coupled water flux is the underlying mechanism of the corneal endothelial pump, which is highly dependent on the presence of bicarbonate. In this study we test the hypothesis that the increased intracellular pH (pHi) caused by bicarbonate stimulates glycolytic activity and the production of lactate by endothelial cells. Primary cultures of bovine corneal endothelial cells (BCEC) were incubated in bicarbonate-free (BF) ringer, a high [HEPES] ringer, and bicarbonate-rich (BR) ringer all at pH 7.5. Lactate production and glucose consumption were greatest in BR>HEPES >BF. Similarly, pHi was greatest in BR>HEPES>BF. Increasing pHi with NH4Cl also increased lactate production in BF or BR, indicating that the increased lactate production in BR is not due to HCO3- itself. Glucose transport capacity, as measured by 2-N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino-2-Deoxyglucose (2-NBDG) uptake was unaffected by the three incubation conditions. Using Laconic, a FRET sensor for lactate, we found that intracellular [lactate] increased immediately and transiently when cells were switched from BF to BR perfusion indicating increased lactate production with subsequent matching of efflux. Moreover, induction of acute lactate influx by perfusion pulses of 10 mM lactate increased intracellular [lactate] significantly faster in BF than in BR, consistent with higher lactate production and efflux in BR. In summary, our results indicate that glycolytic flux and lactate production increase in BR due to increased pHi, consistent with the well-known pH sensitivity of phosphofructokinase, the rate limiting enzyme in glycolysis.

Ammonia sensitive SLC4A11 mitochondrial uncoupling reduces glutamine induced oxidative stress.

SLC4A11 is a NH3 sensitive membrane transporter with H+ channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown. Here we demonstrate that SLC4A11 is localized to the inner mitochondrial membrane of CE and SLC4A11 transfected PS120 fibroblasts, where it acts as an NH3-sensitive mitochondrial uncoupler that enhances glutamine-dependent oxygen consumption, electron transport chain activity, and ATP levels by suppressing damaging Reactive Oxygen Species (ROS) production. In the presence of glutamine, Slc4a11-/- (KO) mouse CE generate significantly greater mitochondrial superoxide, a greater proportion of damaged depolarized mitochondria, and more apoptotic cells than WT. KO CE can be rescued by MitoQ, reducing NH3 production by GLS1 inhibition or dimethyl αKetoglutarate supplementation, or by BAM15 mitochondrial uncoupling. Slc4a11 KO mouse corneal edema can be partially reversed by αKetoglutarate eye drops. Moreover, we demonstrate that this role for SLC4A11 is not specific to CE cells, as SLC4A11 knockdown in glutamine-addicted colon carcinoma cells reduced glutamine catabolism, increased ROS production, and inhibited cell proliferation. Overall, our studies reveal a unique metabolic mechanism that reduces mitochondrial oxidative stress while promoting glutamine catabolism.

RPE65 has an additional function as the lutein to meso-zeaxanthin isomerase in the vertebrate eye.

Carotenoids are plant-derived pigment molecules that vertebrates cannot synthesize de novo that protect the fovea of the primate retina from oxidative stress and light damage. meso-Zeaxanthin is an ocular-specific carotenoid for which there are no common dietary sources. It is one of the three major carotenoids present at the foveal center, but the mechanism by which it is produced in the eye is unknown. An isomerase enzyme is thought to be responsible for the transformation of lutein to meso-zeaxanthin by a double-bond shift mechanism, but its identity has been elusive. We previously found that meso-zeaxanthin is produced in a developmentally regulated manner in chicken embryonic retinal pigment epithelium (RPE)/choroid in the absence of light. In the present study, we show that RPE65, the isomerohydrolase enzyme of the vertebrate visual cycle that catalyzes the isomerization of all-trans-retinyl esters to 11-cis-retinol, is also the isomerase enzyme responsible for the production of meso-zeaxanthin in vertebrates. Its RNA is up-regulated 23-fold at the time of meso-zeaxanthin production during chicken eye development, and we present evidence that overexpression of either chicken or human RPE65 in cell culture leads to the production of meso-zeaxanthin from lutein. Pharmacologic inhibition of RPE65 function resulted in significant inhibition of meso-zeaxanthin biosynthesis during chicken eye development. Structural docking experiments revealed that the epsilon ring of lutein fits into the active site of RPE65 close to the nonheme iron center. This report describes a previously unrecognized additional activity of RPE65 in ocular carotenoid metabolism.

All three human scavenger receptor class B proteins can bind and transport all three macular xanthophyll carotenoids.

Carotenoids are plant pigment molecules that are potent antioxidants. Carotenoids cannot be synthesized de novo; therefore, their dietary intake and transport to various tissues are essential to harness their health benefits. Two of the three scavenger receptor class B (SRB) proteins, SR-B1 and CD36, have been implicated as carotenoid transporters in lower species and in various tissues of higher animals. The function of the third SRB protein, SR-B2, in carotenoid transport is unknown. Using surface plasmon resonance (SPR) analyses, we have determined that all three human SRB proteins are capable of binding the macular xanthophyll carotenoids; lutein, zeaxanthin, and meso-zeaxanthin. By over-expressing human SRB proteins in cells that do not endogenously express SRBs, we have determined that lutein uptake is enhanced in the presence of LDL and is mediated by SR-B1 and CD36. SR-B1, SR-B2, and CD36 were able to take up significant amounts of zeaxanthin as well as meso-zeaxanthin, and uptake was increased in the presence of HDL. Our analyses revealed no apparent differences in protein expression profiles of SRBs in central and peripheral regions of human donor tissues, indicating that carotenoid-binding proteins rather than transporters are likely to mediate selective accumulation of carotenoids into the macula.

Developmentally Regulated Production of meso-Zeaxanthin in Chicken Retinal Pigment Epithelium/Choroid and Retina.

PURPOSE: meso-Zeaxanthin is a carotenoid that is rarely encountered in nature outside of the vertebrate eye. It is not a constituent of a normal human diet, yet this carotenoid comprises one-third of the primate macular pigment. In the current study, we undertook a systematic approach to biochemically characterize the production of meso-zeaxanthin in the vertebrate eye. METHODS: Fertilized White Leghorn chicken eggs were analyzed for the presence of carotenoids during development. Yolk, liver, brain, serum, retina, and RPE/choroid were isolated, and carotenoids were extracted. The samples were analyzed on C-30 or chiral HPLC columns to determine the carotenoid composition. RESULTS: Lutein and zeaxanthin were found in all studied nonocular tissues, but no meso-zeaxanthin was ever detected. Among the ocular tissues, the presence of meso-zeaxanthin was consistently observed starting at embryonic day 17 (E17) in the RPE/choroid, several days before its consistent detection in the retina. If RPE/choroid of an embryo was devoid of meso-zeaxanthin, the corresponding retina was always negative as well. CONCLUSIONS: This is the first report of developmentally regulated synthesis of meso-zeaxanthin in a vertebrate system. Our observations suggest that the RPE/choroid is the primary site of meso-zeaxanthin synthesis. Identification of meso-zeaxanthin isomerase enzyme in the developing chicken embryo will facilitate our ability to determine the biochemical mechanisms responsible for production of this unique carotenoid in other higher vertebrates, such as humans.

Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease.

The human macula uniquely concentrates three carotenoids: lutein, zeaxanthin, and meso-zeaxanthin. Lutein and zeaxanthin must be obtained from dietary sources such as green leafy vegetables and orange and yellow fruits and vegetables, while meso-zeaxanthin is rarely found in diet and is believed to be formed at the macula by metabolic transformations of ingested carotenoids. Epidemiological studies and large-scale clinical trials such as AREDS2 have brought attention to the potential ocular health and functional benefits of these three xanthophyll carotenoids consumed through the diet or supplements, but the basic science and clinical research underlying recommendations for nutritional interventions against age-related macular degeneration and other eye diseases are underappreciated by clinicians and vision researchers alike. In this review article, we first examine the chemistry, biochemistry, biophysics, and physiology of these yellow pigments that are specifically concentrated in the macula lutea through the means of high-affinity binding proteins and specialized transport and metabolic proteins where they play important roles as short-wavelength (blue) light-absorbers and localized, efficient antioxidants in a region at high risk for light-induced oxidative stress. Next, we turn to clinical evidence supporting functional benefits of these carotenoids in normal eyes and for their potential protective actions against ocular disease from infancy to old age.

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.

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.

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 ß5 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 Glu(50) 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 Leu(157) 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.

Wnt gene expression in human trabecular meshwork cells.

PURPOSE: The aim of this study was to examine the expression of genes related to the Wnt signaling pathway, such as beta-catenin (CTNNB1) and secreted frizzled-related protein-1 (sFRP1), in human trabecular meshwork (TM) cells. In addition, the effect of oxidative stress on Wnt signaling was evaluated. METHODS: All experiments were conducted using second- or third-passaged human TM cells. cDNA was prepared from total RNA extracted from cells by means of reverse transcription. PCR was then performed to determine the presence of Wnt genes. For oxidative stress, TM cells were treated with 1 mM of H(2)O(2) for 30 min. Actin staining was carried out to verify cell response to oxidative stress. Western blotting was used to measure Wnt-related protein levels after H(2)O(2) treatment. RESULTS: Positive PCR products were detected for a total of 25 Wnt and Wnt-related genes in human TM cells. Most of the genes identified belonged to the Wnt/beta-catenin pathway. Members of the beta-catenin-independent noncanonical pathways were also found. Oxidative stress did not result in significant changes in beta-catenin and sFRP1 protein levels. CONCLUSIONS: Genes related to canonical and noncanonical Wnt pathways are expressed in human TM cells. It appears that all three Wnt pathways are operative in the TM system. Oxidative stress, while thought to play a role in the development of glaucoma, had little effect on the Wnt activity in TM cells.

Differential effects of myocilin and optineurin, two glaucoma genes, on neurite outgrowth.

Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOC(WT)-EGFP, pMYOC(P370L)-EGFP, pMYOC(1-367)-EGFP, pOPTN(WT)-EGFP, and pOPTN(E50K)-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTN(WT)-EGFP and pOPTN(E50K)-EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.