Browsing by Author "Patel, Pinkal D."

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ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load
(Springer Nature, 2020-11-05) Kasetti, Ramesh B.; Patel, Pinkal D.; Maddineni, Prabhavathi; Patil, Shruti; Kiehlbauch, Charles; Millar, J. Cameron; Searby, Charles C.; Raghunathan, Vijaykrishna; Sheffield, Val C.; Zode, Gulab S.
The underlying pathological mechanisms of glaucomatous trabecular meshwork (TM) damage and elevation of intraocular pressure (IOP) are poorly understood. Here, we report that the chronic endoplasmic reticulum (ER) stress-induced ATF4-CHOP-GADD34 pathway is activated in TM of human and mouse glaucoma. Expression of ATF4 in TM promotes aberrant protein synthesis and ER client protein load, leading to TM dysfunction and cell death. These events lead to IOP elevation and glaucomatous neurodegeneration. ATF4 interacts with CHOP and this interaction is essential for IOP elevation. Notably, genetic depletion or pharmacological inhibition of ATF4-CHOP-GADD34 pathway prevents TM cell death and rescues mouse models of glaucoma by reducing protein synthesis and ER client protein load in TM cells. Importantly, glaucomatous TM cells exhibit significantly increased protein synthesis along with induction of ATF4-CHOP-GADD34 pathway. These studies indicate a pathological role of ATF4-CHOP-GADD34 pathway in glaucoma and provide a possible treatment for glaucoma by targeting this pathway.
CNS axonal degeneration and transport deficits at the optic nerve head precede structural and functional loss of retinal ganglion cells in a mouse model of glaucoma
(BioMed Central Ltd., 2020-08-27) Maddineni, Prabhavathi; Kasetti, Ramesh B.; Patel, Pinkal D.; Millar, J. Cameron; Kiehlbauch, Charles; Clark, Abbot F.; Zode, Gulab S.
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.
Phenotypic and transcriptomic comparison of genetically distinct mouse strains for susceptibility to glucocorticoid-induced ocular hypertension (GC-OHT)
(2024-03-21) Patel, Pinkal D.; Millar, J. Cameron; Curry, Stacy; Feris, Sherri; Clark, Abbot F.
Purpose: Anti-inflammatory and immunosuppressive glucocorticoids (GCs) are widely prescribed for a variety of conditions and diseases. Unfortunately, a significant number of people experience negative side-effects associated with long term GC therapy and develop GC-induced ocular hypertension (GC-OHT) leading to secondary glaucoma. GC-OHT shares clinical and molecular signatures with primary open angle glaucoma (POAG) making this an appropriate model to study POAG. However, not all humans develop GC-OHT when treated with GCs. The ones that develop GC-OHT are called ‘responders’ whereas the ones that do not respond to GCs are called ‘non-responders’. The purpose of our study is to: (1) determine whether there are mouse strain differences in the development of GC-OHT, (2) whether resistance to develop GC-OHT is correlated with endogenous TM tissue gene expression using transcriptomic analysis. Methods: After measurement of baseline IOP, various mouse strains (B6, D2.gpnmb⁺, BALB/cJ, 129P3/J, C3H/HeJ) were treated with weekly periocular injections of potent GC dexamethasone (DEX; n=5-10) or vehicle (n=5-10) in both eyes for 4-5 weeks. IOPs were measured weekly using a TonoLab rebound tonometer in isoflurane anesthetized mice. “TM ring” tissue and underlying sclera was carefully collected, and mRNA libraries were prepared for sequencing. Differential expression analysis was performed to identify DEX-induced changes within each strain. Furthermore, Ingenuity Pathway Analysis (IPA) was used to identify DEX-altered pathways in each strain and compare differences between responder and non-responder strains. Results: B6 and C3H/HeJ mice robustly and reproducibly develop DEX-OHT with ΔIOP of 5-8 mmHg (P<0.0001). In contrast, D2.gpnmb⁺, 129P3/J, and BALB/cJ mice were resistant to the development of DEX-OHT. Differential analysis of gene expression between mouse strains showed novel DEX-responsive genes in all strains. Moreover, comparison of mouse strains using IPA showed similarities in the pathway and networks of the responder strains (B6 and C3H/HeJ). Conclusions: As observed in humans, we find that there are differences in GC responsiveness and the ability to develop GC-OHT among mouse strains. Transcriptomics evidence suggests that responder strains share common pathways that contribute towards development of GC-OHT. These studies will reveal the molecular mechanisms responsible for GC-OHT as well as provide insights into the pathogenesis of POAG.
Role of Glucocorticoids and Glucocorticoid Receptors in Glaucoma Pathogenesis
(MDPI, 2023-10-27) Patel, Pinkal D.; Kodati, Bindu; Clark, Abbot F.
The glucocorticoid receptor (GR), including both alternative spliced isoforms (GRalpha and GRbeta), 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 GRbeta, 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 GRbeta can block GC-induced pathogenic changes in TM cells and reverse GC-OHT in mice. The endogenous expression of GRbeta 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.
The Role of Mechanosensory TRPV4 Channels and Nitric Oxide Signaling in Intraocular Pressure Homeostasis and its Impairment in Glaucoma
(2020-08) Patel, Pinkal D.; Zode, Gulab S.; Clark, Abbot F.; Pang, Iok-Hou; Krishnamoorthy, Raghu R.; Rickards, Caroline A.
Several population-based studies have identified elevated intraocular pressure (IOP) as a major causative risk factor associated with primary open angle glaucoma (POAG), the most common form of glaucoma that affects millions of people worldwide. Moreover, multi-ethnic clinical trials in several different countries over the last few decades have provided overwhelming evidence showing correlation between lowering of IOP and reduced progression of vision loss. As a result, IOP reducing therapeutic interventions are the gold standard in glaucoma therapy. Although the role of IOP is evident in pathology of POAG, very few studies have delved into the complex physiological mechanisms that regulate IOP homeostasis. From continuous telemetry recordings in nonhuman primates, we now know that IOP is a dynamic variable that fluctuates throughout the day. However, despite the fluctuations, the mean IOP is still maintained within a narrow physiological range. The level of IOP elevation at any given time depends on the resistance to aqueous humor outflow encountered in the conventional outflow pathway consisting of the trabecular meshwork (TM), Schlemm's canal (SC), and the distal episcleral vessels. Recent studies have suggested that the cells of the outflow pathway have intrinsic ability to detect biomechanical stimuli in their environment (like shear stress) and convert these stimuli into biochemical signals to elicit specific cellular responses. Although mechanotransduction at the TM is deemed critical for IOP homeostasis, we are yet to conclusively identify the exact signaling pathway involved. In this study, we identify the role of transient receptor potential vanilloid IV channels (TRPV4) in sensing mechanical stress on the TM. We show that shear stress activates TRPV4 channels in human primary TM cells, which leads to endothelial nitric oxide synthase (eNOS)-dependent nitric oxide (NO) production. NO, itself has been identified as a key regulator of IOP. Exogenous NO delivery to the eye has been shown to reduce IOP in humans. However, the underlying mechanism that regulates endogenous levels of NO still remains unknown. To this end, we demonstrate that TRPV4 channels regulate eNOS-dependent NO production in primary human TM cells and ex vivo cultured human TM tissues. We show that TRPV4 activation by mechanical shear leads to activation of eNOS signaling and NO production. Furthermore, pharmacological activation of TRPV4 channels via a selective agonist GSK1016790A (GSK101) leads to eNOS phosphorylation and NO production. In animal models, we demonstrate a role of TRPV4 channels in regulating physiological IOP. Treatment of C57BL/6J mouse eyes with TRPV4 agonist GSK101 leads to reduction in baseline night-time IOP and nominal improvement in outflow facility. We also show that conditional knockout of TRPV4 channels in Ad5-Cre injected TRPV4f/f mice leads to increase in IOP. We use the NOS3-/- (eNOS) to further show that TRPV4 mediated lowering of IOP is eNOS dependent. Dysregulation of the TM cells leads to increase in resistance and IOP elevation. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in lowering the resting IOP. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.