Ramesh B. Kasetti

CRISPR-Cas9–based treatment of myocilin-associated glaucoma

Significance A mutation in myocilin is the most common known genetic cause of primary open-angle glaucoma (POAG). These mutations, which are dominant in nature, affect trabecular meshwork (TM) health and/or function and cause elevated intraocular pressure. Using in vitro human trabecular meshwork cells, an in vivo mouse model, and ex vivo human eyes, our study demonstrates the potential of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing in human myocilin-associated POAG. By disrupting the mutant myocilin gene and its function using CRISPR-Cas9, we were able to reduce associated endoplasmic reticulum stress, lower intraocular pressure, and prevent further glaucomatous damage in mouse eyes. We also show the feasibility of using the CRISPR-Cas9 system in cultured human eyes. Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, with elevated intraocular pressure (IOP) a major risk factor. Myocilin (MYOC) dominant gain-of-function mutations have been reported in ∼4% of POAG cases. MYOC mutations result in protein misfolding, leading to endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that regulates IOP. We use CRISPR-Cas9–mediated genome editing in cultured human TM cells and in a MYOC mouse model of POAG to knock down expression of mutant MYOC, resulting in relief of ER stress. In vivo genome editing results in lower IOP and prevents further glaucomatous damage. Importantly, using an ex vivo human organ culture system, we demonstrate the feasibility of human genome editing in the eye for this important disease.

Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork

Purpose Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. Methods Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. Results Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 μl/min/mm Hg in Tg-MYOCY437H vs. 0.0332 μl/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. Conclusions These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.

Dexamethasone-Induced Ocular Hypertension in Mice: Effects of Myocilin and Route of Administration

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious adverse effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. An appropriate mouse model can help us understand precise molecular mechanisms and etiology of GC-induced OHT. We therefore developed a novel, simple, and reproducible mouse model of GC-induced OHT. GC-induced myocilin expression in the trabecular meshwork (TM) has been suggested to play an important role in GC-induced OHT. We further determined whether myocilin contributes to GC-OHT. C57BL/6J mice received weekly periocular conjunctival fornix injections of a dexamethasone-21-acetate (DEX-Ac) formulation. Intraocular pressure (IOP) elevation was relatively rapid and significant, and correlated with reduced conventional outflow facility. Nighttime IOPs were higher in ocular hypertensive eyes compared to daytime IOPs. DEX-Ac treatment led to increased expression of fibronectin, collagen I, and α-smooth muscle actin in the TM in mouse eyes. No changes in body weight indicated no systemic toxicity associated with DEX-Ac treatment. Wild-type mice showed increased myocilin expression in the TM on DEX-Ac treatment. Both wild-type and Myoc-/- mice had equivalent and significantly elevated IOP with DEX-Ac treatment every week. In conclusion, our mouse model mimics many aspects of GC-induced OHT in humans, and we further demonstrate that myocilin does not play a major role in DEX-induced OHT in mice.

Study of corneal epithelial progenitor origin and the Yap1 requirement using keratin 12 lineage tracing transgenic mice

Key issues in corneal epithelium biology are the mechanism for corneal epithelium stem cells to maintain the corneal epithelial homeostasis and wound healing responses, and what are the regulatory molecular pathways involved. There are apparent discrepancies about the locations of the progenitor populations responsible for corneal epithelial self-renewal. We have developed a genetic mouse model to trace the corneal epithelial progenitor lineages during adult corneal epithelial homeostasis and wound healing response. Our data revealed that the early corneal epithelial progenitor cells expressing keratin-12 originated from limbus, and gave rise to the transit amplifying cells that migrated centripetally to differentiate into corneal epithelial cells. Our results support a model that both corneal epithelial homeostasis and wound healing are mainly maintained by the activated limbal stem cells originating form limbus, but not from the corneal basal epithelial layer. In the present study, we further demonstrated the nuclear expression of transcriptional coactivator YAP1 in the limbal and corneal basal epithelial cells and its essential role for maintaining the high proliferative potential of those corneal epithelial progenitor cells in vivo.

Increased synthesis and deposition of extracellular matrix proteins leads to endoplasmic reticulum stress in the trabecular meshwork

Increased synthesis and deposition of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) is associated with TM dysfunction and intraocular pressure (IOP) elevation in glaucoma. However, it is not understood how ECM accumulation leads to TM dysfunction and IOP elevation. Using a mouse model of glucocorticoid (GC)-induced glaucoma, primary human TM cells and human post-mortem TM tissues, we show that increased ECM accumulation leads to endoplasmic reticulum (ER) stress in the TM. The potent GC, dexamethasone (Dex) increased the secretory protein load of ECM proteins in the ER of TM cells, inducing ER stress. Reduction of fibronectin, a major regulator of ECM structure, prevented ER stress in Dex-treated TM cells. Overexpression of fibronectin via treatment with cellular fibronectin also induced chronic ER stress in primary human TM cells. Primary human TM cells grown on ECM derived from Dex-treated TM cells induced ER stress markers. TM cells were more prone to ER stress from ECM accumulation compared to other ocular cell types. Moreover, increased co-localization of ECM proteins with ER stress markers was observed in human post-mortem glaucomatous TM tissues. These data indicate that ER stress is associated with increased ECM accumulation in mouse and human glaucomatous TM tissues.

Methods for Analyzing Endoplasmic Reticulum Stress in the Trabecular Meshwork of Glaucoma Models

The pathological mechanisms underlying increased outflow resistance at the trabecular meshwork (TM) that is responsible for elevating intraocular pressure (IOP) have not been fully delineated. Recent studies have shown that progressive accumulation of misfolded proteins and induction of endoplasmic reticulum (ER) stress is associated with the pathophysiology of glaucomatous TM damage and IOP elevation. We have shown that known causes of human glaucoma, including expression of mutant myocilin or dexamethasone treatment induce abnormal protein accumulation and ER stress in the TM in vitro and in vivo models. To cope up with abnormal protein accumulation, TM cells activate a cytoprotective pathway of unfolded protein response (UPR). However, chronic ER stress can lead to TM dysfunction and IOP elevation. Using cell culture, mouse models, and human postmortem tissues as well as genetic and pharmacological manipulations, we have analyzed ER stress and UPR mediators in the glaucomatous TM damage and IOP elevation. In this chapter, we have described a detailed protocol for the analysis of protein misfolding and ER stress in TM cells and tissues and its association with glaucomatous TM damage and IOP elevation.

Inhibition of Transforming Growth Factor-β2 Signaling Prevents ECM Remodeling, Endoplasmic Reticulum Stress and Ocular Hypertension in Steroid-induced Glaucoma

We thank Wostyn and De Deyn for their excellent response to our recent correspondence ‘‘Choroidal Folds in Astronauts’’ published in IOVS and for their insightful ideas regarding the potential role of the optic nerve sheath (ONS) in the production of disc swelling, globe flattening, and choroidal folds observed during and after long-duration space flight (LDSF). Two basic mechanisms, or perhaps a combination of the two, are hypothesized to account for elevated cerebrospinal fluid (CSF) pressure within the orbital subarachnoid space (SAS) that may be responsible for these anatomic changes. The first mechanism is based on the notion that a rise in intracranial pressure (ICP) occurs during space flight as a result of a microgravity (MG)-induced cephalad fluid shift that produces venous stasis in the head and neck. This stasis may lead to impairment of CSF outflow from the brain, cerebral venous congestion, and a resultant increase in ICP which may then be transmitted down the ONSs to the intraorbital SAS. The second mechanism proposes that these changes may result from a local, MG-induced rise in ONS pressure within the orbit with or without a rise in ICP. The intracanalicular portion of the optic nerve in this setting might be a ‘‘bottleneck,’’ leading to reduced outflow of CSF from the distended ONS. A similar mechanism may occur in terrestrial intracanalicular or posterior orbital apex nerve tumors (e.g., sheath meningiomas) where the visual acuity remains normal, the visual field might show only an enlarged blind spot, and the optic disc may show chronic edema for years without the development of optic atrophy or significant visual loss over time. The end result of either mechanism is a rise in CSF pressure within the SAS of the orbit that may cause ONS distention and a concurrent anteriorly directed force that indents the posterior globe. As the authors point out, this posterior globe flattening would account for the axial shortening, hyperopic shift, and choroidal folds documented in many astronauts. Additionally, this same increase in ONS pressure could compress the ON within the orbit and, in conjunction with its effects across the lamina cribrosa, may produce stasis of axoplasmic flow with resultant optic disc swelling. It should be noted that ON sheath expansion and globe flattening have been documented by inflight ultrasound only 10 days into a space mission. Therefore, whatever the specific mechanism, these anatomic changes occur rather quickly following exposure to MG. Furthermore, posterior globe flattening has been documented for more than 7 years following LDSF suggesting that these changes may be permanent (C. Robert Gibson, personal communication, 2017). It is possible that increased ONS pressure may act in conjunction with metabolic toxins within the ONS to structurally remodel the posterior sclera. We would also like to mention that the National Aeronautics and Space Administration has recently changed the nomenclature for this syndrome from visual impairment intracranial pressure syndrome, as stated by the authors, to space flight–associated neuro-ocular syndrome (SANS) as this is thought to be a more appropriate descriptive term. The authors propose that the ONS response to the rise in CSF pressure within the orbital SAS may impact the susceptibility of an astronaut to the anatomic changes of SANS. This ONS response to pressure could indeed help to explain the large spectrum of anatomic change noted in astronauts during and after nearly identical MG exposure on the ISS. They point out the work of Hansen and Helmke, who used intrathecal infusion to demonstrate that the extent of ONS dilation, as measured by ultrasonography, was directly correlated with increasing CSF pressure within the sheath until a saturation point was reached at which no further dilation occurred. Presumably, at this saturation point, the ONS assumes a degree of rigidity that prevents further expansion and causes a more direct transfer of increased CSF pressure to the posterior globe and ON. The authors point out that as this compensatory mechanism reaches its limit even small increases in CSF volume may result in prominent increases in CSF pressure within the ONS. Therefore, a low saturation point may anatomically predispose an astronaut to a more prominent increase in pressure. In contrast, a degree of protection may be associated with a higher saturation point. In this scenario, sufficient ONS elasticity may result in continued expansion of the ONS with less of a SAS pressure increase. Thus, variations in elasticity within the structure of the ONS may result in dissimilar degrees of anatomic change to the globe and ON during spaceflight. Hansen et al. also documented that following ONS dilation there can be a permanent expansion or resetting of the ONS. The long-term persistence of varying degrees of post mission globe flattening and associated refractive changes in astronauts during MG exposure suggests a similar permanent resetting of the posterior globe contour that may be partially determined by the elastic properties of the ONS. Further work is necessary and ongoing to determine the basic mechanism causing SANS as we prepare for possible longer duration space flights including a potential manned mission to Mars.