Molecular Mechanisms Of Glaucoma

National Eye Institute

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Abstract

Several approaches for RGC neuroprotection have been suggested and tested in animal models. We previously demonstrated that intravitreal injection of human bone marrow-derived mesenchymal stem cells (BMSCs), or small extracellular vesicles (sEVs) secreted by BMSCs, provided statistically significant RGC neuroprotection when compared to control samples. Our data also revealed that miRNAs play an important role in the observed RGC neuroprotection. The emerging approach of sEV-based RGC protection demands more comprehensive investigation. sEVs isolated using traditional methods represent a heterogeneous population of sEVs which are comprised of different subpopulations such as microvesicles, exosomes, and recently identified mitovesicles. Hence, we fractionated isolated sEVs using density gradient-based differential ultracentrifugation and compared their neuroprotective effects with unfractionated sEVs on RGC survival and neurite outgrowth in vitro and in vivo. Using mixed primary mouse retinal cell cultures, we demonstrated that unfractionated and fractionated sEVs (F16-F20 and F11-F20) from conditioned medium (CM) of undifferentiated BRN3B-H9 and NCRM-1 cells provide comparative neuroprotection (≥2-fold); however, F16-F20 and F11-F20 fractions show additional improvement in the morphometry of RGCs. These in vitro effects of sEVs derived from NCRM-1 cells were further validated in vivo using a magnetic bead-induced ocular hypertension model of glaucoma in C57BL/6J mice after 60 days of sEV intravitreal administration. The proportion of surviving RGCs (Rbpms- and Brn3a-positive cells) in the peripheral, mid-peripheral, and central regions of the retina was comparable between unfractionated and fractionated (F16–F20) sEV-treated groups, and significantly higher than in untreated retinas. The most pronounced neuroprotection after sEVs administration was observed in peripheral retina followed by mid-peripheral, and central regions. These findings highlight the therapeutic potential of sEV-based interventions in mitigating glaucomatous neurodegeneration. Comparison of differentially expressed genes between fractionated and unfractionated sEVs revealed a significant enrichment of energy-related pathways in the fractionated samples, which may underlie the enhanced neurite outgrowth of RGCs observed in our in vitro neuroprotection study. To further identify the specific mechanism responsible for the observed neuroprotective effects, we developed an in vitro model of human retinal ganglion-like cells (RGLCs, derived from BRN3B-H9 cells) by inhibiting the mitochondrial electron transport chain using sodium azide. Our findings indicate that fractionated sEVs preserve RGC morphometry more effectively than unfractionated sEVs, potentially by promoting metabolic reprogramming through enhanced glycolysis and/or reduction of mitochondrial stress and reactive oxygen species. Thus, fractionated sEVs can help RGCs survive sodium azide toxicity by shifting metabolism away from damaged mitochondria toward glycolysis, which is a well-known protective response under mitochondrial inhibition. These protective effects are likely mediated by bioactive cargo within the sEVs, including miRNAs, mRNAs, lncRNAs, and proteins, which modulate intracellular pathways associated with energy metabolism and cellular resilience. To identify these key cargo molecules and their downstream targets, ongoing transcriptomic, proteomic, and metabolomic profiling of sEV-treated RGLCs is underway. These comprehensive datasets, combined with comparisons across sEVs derived from different cell types, aim to uncover cell-specific therapeutic candidates and mechanisms of neuroprotective effects. These findings are also being analyzed for comparison of candidates among different set of sEVs derived from different cell types. Additionally, to study the effects of sEVs on RGCs, we tested different types of chemical-induced stress including oxidative stress (hydrogen peroxide), microtubule destabilization (colchicine), hypoxia (cobalt chloride), and ATP depletion, and elevated reactive oxygen species levels in RGLCs. These models provide a controlled in vitro platform to evaluate the protective or restorative effects of sEVs (including fractionated vs. unfractionated sEVs from different sources) under specific stress conditions that reflect disease-relevant cellular dysfunctions. They allow for the investigation of molecular and cellular pathways modulated by sEVs, including oxidative stress response, mitochondrial dynamics, apoptosis/necrosis pathways, and metabolic reprogramming. Hence, these chemical-induced stress models in human RGLCs will be used to simulate glaucoma-relevant cellular damage and evaluate the neuroprotective and mechanistic effects of sEV-based therapies. To further investigate comprehensive transcriptomic changes in specific retinal cell types following sEV treatment in glaucomatous retina, we have initiated experiments for Xenium-HD Spatial Transcriptomics using a custom add-on panel of 50 genes to identify RGC subtype-specific molecular changes in fresh-frozen retinal tissue sections in an ocular hypertension mouse model. By integrating fluorescence histology with high-resolution, spatially resolved gene expression profiling, this approach will enable us to gain unprecedented insights into tissue architecture and cellular function at the single-cell resolution. This approach holds significant potential for identifying spatial gene expression patterns and molecular signatures associated with sEV-mediated neuroprotection. Collectively, our findings provide critical insights into the functional components of sEVs responsible for neuroprotection and lay the groundwork for future translational strategies. These findings could facilitate the development of targeted sEV formulations tailored to specific retinal cell types or disease stages, ultimately guiding personalized therapeutic strategies for glaucoma and other neurodegenerative retinal diseases.