Regulation of Diseases-Associated Microglia in the Optic Nerve by Lipoxin B4 and Ocular Hypertension.

Background: The resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B4 produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB4 neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap. Methods: Cellular targets and signaling of LXB4 in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of the mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB4 in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups was assessed by Student's t-test and one-way ANOVA. Results: Single-cell transcriptomics identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB4 treatment. Conclusion: We identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB4 target in the retina and optic nerve. LXB4 maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB4 pathway.

Dysregulation of neuroprotective lipoxin pathway in astrocytes in response to cytokines and ocular hypertension​.

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte reactivity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension. By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4's neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.

Dysregulation of Neuroprotective Lipoxin Pathway in Astrocytes in Response to Cytokines and Ocular Hypertension.

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte activity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension (n=40). By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4's neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.

Role of vasorin, an anti-apoptotic, anti-TGF-ß and hypoxia-induced glycoprotein in the trabecular meshwork cells and glaucoma.

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open-angle glaucoma patients compared to non-glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti-apoptotic and anti-TGF-ß activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF-ß2-induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride-induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF-α-induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF-ß induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.

Elevated Levels of Growth/Differentiation Factor-15 in the Aqueous Humor and Serum of Glaucoma Patients.

Dysregulated levels of growth/differentiation factor-15 (GDF15), a divergent member of the transforming growth factor-beta super family, have been found to be associated with the pathology of various diseases. In this study, we evaluated the levels of GDF15 in aqueous humor (AH) and serum samples derived from primary open-angle glaucoma (POAG) and age- and gender-matched non-glaucoma (cataract) patients to assess the plausible association between GDF15 and POAG. GDF15 levels were determined using an enzyme-linked immunosorbent assay, and data analysis was performed using the Wilcoxon rank sum test, or the Kruskal-Wallis test and linear regression. GDF15 levels in the AH (n = 105) of POAG patients were significantly elevated (by 7.4-fold) compared to cataract patients (n = 117). Serum samples obtained from a subgroup of POAG patients (n = 41) also showed a significant increase in GDF15 levels (by 50%) compared to cataract patients. GDF15 levels were elevated in male, female, African American, and Caucasian POAG patients. This study reveals a significant and marked elevation of GDF15 levels in the AH of POAG patients compared to non-glaucoma cataract control patients. Although serum GDF15 levels were also elevated in POAG patients, the magnitude of difference was much smaller relative to that found in the AH.

Drebrin, an actin-binding protein, is required for lens morphogenesis and growth.

BACKGROUND: Lens morphogenesis, architecture, and clarity are known to be critically dependent on actin cytoskeleton organization and cell adhesive interactions. There is limited knowledge, however regarding the identity and role of key proteins regulating actin cytoskeletal organization in the lens. This study investigated the role of drebrin, a developmentally regulated actin-binding protein, in mouse lens development by generating and characterizing a conditional knockout (cKO) mouse model using the Cre-LoxP recombination approach. RESULTS: Drebrin E, a splice variant of DBN1 is a predominant isoform expressed in the mouse lens and exhibits a maturation-dependent downregulation. Drebrin co-distributes with actin in both epithelium and fibers. Conditional deficiency (both haploinsufficiency and complete absence) of drebrin results in disrupted lens morphogenesis leading to cataract and microphthalmia. The drebrin cKO lens reveals a dramatic decrease in epithelial height and width, E-cadherin, and proliferation, and increased apoptotic cell death and expression of α-smooth muscle actin, together with severely impaired fiber cell organization, polarity, and cell-cell adhesion. CONCLUSIONS: This study demonstrates the requirement of drebrin in lens development and growth, with drebrin deficiency leading to impaired lens morphogenesis and microphthalmia.

Biochemical and biomechanical characteristics of dystrophin-deficient mdx3cv mouse lens.

The molecular and cellular basis for cataract development in mice lacking dystrophin, a scaffolding protein that links the cytoskeleton to the extracellular matrix, is poorly understood. In this study, we characterized lenses derived from the dystrophin-deficient mdx3cv mouse model. Expression of Dp71, a predominant isoform of dystrophin in the lens, was induced during lens fiber cell differentiation. Dp71 was found to co-distribute with dystroglycan, connexin-50 and 46, aquaporin-0, and NrCAM as a large cluster at the center of long arms of the hexagonal fibers. Although mdx3cv mouse lenses exhibited dramatically reduced levels of Dp71, only older lenses revealed punctate nuclear opacities compared to littermate wild type (WT) lenses. The levels of dystroglycan, syntrophin, and dystrobrevin which comprise the dystrophin-associated protein complex (DAPC), and NrCAM, connexin-50, and aquaporin-0, were significantly lower in the lens membrane fraction of adult mdx3cv mice compared to WT mice. Additionally, decreases were observed in myosin light chain phosphorylation and lens stiffness together with a significant elevation in the levels of utrophin, a functional homolog of dystrophin in mdx3cv mouse lenses compared to WT lenses. The levels of perlecan and laminin (ligands of α-dystroglycan) remained normal in dystrophin-deficient lens fibers. Taken together, although mdx3cv mouse lenses exhibit only minor defects in lens clarity possibly due to a compensatory increase in utrophin, the noted disruptions of DAPC, stability, and organization of membrane integral proteins of fibers, and stiffness of mdx3cv lenses reveal the importance of dystrophin and DAPC in maintaining lens clarity and function.