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The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student's t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.

PURPOSE:Fibrosis and a hypoxic environment are associated with the trabecular meshwork (TM) region in the blinding disease glaucoma. Hypoxia has been shown to alter DNA methylation, an epigenetic mechanism involved in regulating gene expression such as the pro-fibrotic transforming growth factor (TGF) β1 and the anti-fibrotic Ras protein activator like 1 (RASAL1). The purpose of this study was to compare DNA methylation levels, and the expression of TGFβ1 and RASAL1 in primary human normal (NTM) with glaucomatous (GTM) cells and in NTM cells under hypoxic conditions. METHODS:Global DNA methylation was assessed by ELISA in cultured age-matched NTM and GTM cells. qPCR was conducted for TGFβ1, collagen 1α1 (COL1A1), and RASAL1 expression. Western immunoblotting was used to determine protein expression. For hypoxia experiments, NTM cells were cultured in a 1%O2, 5%CO2 and 37°C environment. NTM and GTM cells were treated with TGFβ1 (10ng/ml) and the methylation inhibitor 5-azacytidine (5-aza) (0.5μM) respectively to determine their effects on DNA Methyltransferase 1 (DNMT1) and RASAL1 expression. RESULTS:We found increased DNA methylation, increased TGFβ1 expression and decreased RASAL1 expression in GTM cells compared to NTM cells. Similar results were obtained in NTM cells under hypoxic conditions. TGFβ1 treatment increased DNMT1 and COL1A1, and decreased RASAL1 expression in NTM cells. 5-aza treatment decreased DNMT1, TGFβ1 and COL1A1 expression, and increased RASAL1 expression in GTM cells. CONCLUSIONS:TGFβ1 and RASAL1 expression, global DNA methylation, and expression of associated methylation enzymes were altered between NTM and GTM cells. We found that hypoxia in NTM cells induced similar results to the GTM cells. Furthermore, DNA methylation, TGFβ1 and RASAL1 appear to have an interacting relationship that may play a role in driving pro-fibrotic disease progression in the glaucomatous TM.

Abstract Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOC Y437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Glaucoma and age-related macular degeneration (AMD) are the two leading causes of visual loss in the United States. We utilized a novel study design to perform a genome-wide association for both primary open angle glaucoma (POAG) and AMD. This study design utilized a two-stage process for hypothesis generation and validation, in which each disease cohort was utilized as a control for the other. A total of 400 POAG patients and 400 AMD patients were ascertained and genotyped at 500,000 loci. This study identified a novel association of complement component 7 (C7) to POAG. Additionally, an association of central corneal thickness, a known risk factor for POAG, was found to be associated with ribophorin II (RPN2). Linked monogenic loci for POAG and AMD were also evaluated for evidence of association, none of which were found to be significantly associated. However, several yielded putative associations requiring validation. Our data suggest that POAG is more genetically complex than AMD, with no common risk alleles of large effect.

The glucocorticoid receptor (GR), including both alternative spliced isoforms (GRα and GRβ), has been implicated in the development of primary open-angle glaucoma (POAG) and iatrogenic glucocorticoid-induced glaucoma (GIG). POAG is the most common form of glaucoma, which is the leading cause of irreversible vision loss and blindness in the world. Glucocorticoids (GCs) are commonly used therapeutically for ocular and numerous other diseases/conditions. One serious side effect of prolonged GC therapy is the development of iatrogenic secondary ocular hypertension (OHT) and OAG (i.e., GC-induced glaucoma (GIG)) that clinically and pathologically mimics POAG. GC-induced OHT is caused by pathogenic damage to the trabecular meshwork (TM), a tissue involved in regulating aqueous humor outflow and intraocular pressure. TM cells derived from POAG eyes (GTM cells) have a lower expression of GRβ, a dominant negative regulator of GC activity, compared to TM cells from age-matched control eyes. Therefore, GTM cells have a greater pathogenic response to GCs. Almost all POAG patients develop GC-OHT when treated with GCs, in contrast to a GC responder rate of 40% in the normal population. An increased expression of GRβ can block GC-induced pathogenic changes in TM cells and reverse GC-OHT in mice. The endogenous expression of GRβ in the TM may relate to differences in the development of GC-OHT in the normal population. A number of studies have suggested increased levels of endogenous cortisol in POAG patients as well as differences in cortisol metabolism, suggesting that GCs may be involved in the development of POAG. Additional studies are warranted to better understand the molecular mechanisms involved in POAG and GIG in order to develop new disease-modifying therapies to better treat these two sight threatening forms of glaucoma. The purpose of this timely review is to highlight the pathological and clinical features of GC-OHT and GIG, mechanisms responsible for GC responsiveness, potential therapeutic options, as well as to compare the similar features of GIG with POAG.

Abstract Background Glaucoma is a leading neurodegenerative disease affecting over 70 million individuals worldwide. Early pathological events of axonal degeneration and retinopathy in response to elevated intraocular pressure (IOP) are limited and not well-defined due to the lack of appropriate animal models that faithfully replicate all the phenotypes of primary open angle glaucoma (POAG), the most common form of glaucoma. Glucocorticoid (GC)-induced ocular hypertension (OHT) and its associated iatrogenic open-angle glaucoma share many features with POAG. Here, we characterized a novel mouse model of GC-induced OHT for glaucomatous neurodegeneration and further explored early pathological events of axonal degeneration in response to elevated IOP. Methods C57BL/6 J mice were periocularly injected with either vehicle or the potent GC, dexamethasone 21-acetate (Dex) once a week for 10 weeks. Glaucoma phenotypes including IOP, outflow facility, structural and functional loss of retinal ganglion cells (RGCs), optic nerve (ON) degeneration, gliosis, and anterograde axonal transport deficits were examined at various stages of OHT. Results Prolonged treatment with Dex leads to glaucoma in mice similar to POAG patients including IOP elevation due to reduced outflow facility and dysfunction of trabecular meshwork, progressive ON degeneration and structural and functional loss of RGCs. Lowering of IOP rescued Dex-induced ON degeneration and RGC loss, suggesting that glaucomatous neurodegeneration is IOP dependent. Also, Dex-induced neurodegeneration was associated with activation of astrocytes, axonal transport deficits, ON demyelination, mitochondrial accumulation and immune cell infiltration in the optic nerve head (ONH) region. Our studies further show that ON degeneration precedes structural and functional loss of RGCs in Dex-treated mice. Axonal damage and transport deficits initiate at the ONH and progress toward the distal end of ON and target regions in the brain (i.e. superior colliculus). Most of anterograde transport was preserved during initial stages of axonal degeneration (30% loss) and complete transport deficits were only observed at the ONH during later stages of severe axonal degeneration (50% loss). Conclusions These findings indicate that ON degeneration and transport deficits at the ONH precede RGC structural and functional loss and provide a new potential therapeutic window for rescuing neuronal loss and restoring health of damaged axons in glaucoma.

Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is an important factor in the pathogenesis of age-related macular degeneration (AMD). Previous studies have shown that RTA 408, a synthetic triterpenoid compound, potently activates Nrf2. This study aimed to investigate the protective effects of RTA 408 in cultured RPE cells during oxidative stress and to determine the effects of RTA 408 on Nrf2 and its downstream target genes. Primary human RPE cells were pretreated with RTA 408 and then incubated in 200 μM H2O2 for 6 h. Cell viability was measured with the WST-8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining and Hoechst 33342 fluorescent staining. Reduced (GSH) and oxidized glutathione (GSSG) were measured using colorimetric assays. Nrf2 activation and its downstream effects on phase II enzymes were examined by Western blot. Treatment of RPE cells with nanomolar ranges (10 and 100 nM) of RTA 408 markedly attenuated H2O2-induced viability loss and apoptosis. RTA 408 pretreatment significantly protected cells from oxidative stress-induced GSH loss, GSSG formation and decreased ROS production. RTA 408 activated Nrf2 and increased the expression of its downstream genes, such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1. Consequently, the enzyme activities of NQO1, Grx1, and Trx1 were fully protected by RTA 408 pretreatment under oxidative stress. Moreover, knockdown of Nrf2 by siRNA significantly reduced the cytoprotective effects of RTA 408. In conclusion, our data suggest that RTA 408 protect primary human RPE cells from oxidative stress-induced damage by activating Nrf2 and its downstream genes.

Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1–3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death.