Project description:Purpose: Müller glia play dual roles in glaucoma, contributing to both retinal homeostasis and neuroinflammation; their activation by elevated intraocular pressure through the mechanosensitive channel TRPV4 promotes a reactive state that drives retinal ganglion cell (RGC) loss. Lipoxin B4 (LXB4), an endogenous lipid mediator produced by retinal astrocytes, has been shown to suppress glial reactivity and directly protect RGCs. This study investigated whether LXB4 modulates TRPV4-driven Müller glial activation and inflammation and whether Müller glia themselves contribute to this retinal lipoxin pathway. Methods: Ocular hypertension (OHT) was induced in mice via a silicone oil model, and reactive Müller glia were isolated via magnetic sorting for transcriptomic analysis. In vitro, primary and immortalized Müller glia were treated with a TRPV4 agonist with or without LXB4. Glial reactivity was assessed by flow cytometry, immunostaining, qPCR, and western blotting. LC–MS/MS-based lipidomics was used to quantify lipoxin pathway metabolites, and single-cell RNA-seq was used to examine transcriptional responses to LXB4 treatment. GFAP and TRPV4 expression was evaluated via immunohistochemistry in retinal sections. Results: RNA bulk-sequencing analysis and qPCR revealed that Müller glia express both 5- and 15-lipoxygenase. Lipidomic analysis confirmed that the lipoxin pathway is functional and that Müller glia endogenously produce LXB4, establishing this essential cell type as a source of anti-inflammatory and neuroprotective LXB4 in the retina. TRPV4 activation induced a reactive glial phenotype characterized by increased GFAP and IL6 expression, increased STAT3 phosphorylation, and increased production of lipoxin pathway metabolites, suggesting that biomechanical stress simultaneously triggers both gliosis and lipid signaling. Exogenous LXB4 suppressed TRPV4-induced gliosis in vitro by downregulating IL6 and inhibiting STAT3 activation, and in vivo treatment during OHT reduced the expression of Stat3, Il6, and TNF-α while attenuating TRPV4 upregulation in Müller glia. Conclusion: These findings identify a self-regulating lipid circuit in Müller glia and support the targeting of the TRPV4–lipoxin pathway to inhibit gliosis and protect against neurodegeneration in glaucoma.

Project description:Previous studies have demonstrated the dynamic changes in chromatin structure during retinal development that correlate with changes in gene expression. However, a major limitation of those prior studies was the lack of cellular resolution. Here, we integrate single-cell (sc) RNA-seq and scATAC-seq with bulk retinal data sets to identify cell type–specific changes in the chromatin structure during development. Although most genes’ promoter activity is strongly correlated with chromatin accessibility, we discovered several hundred genes that were transcriptionally silent but had accessible chromatin at their promoters. Most of those silent/accessible gene promoters were in the Müller glial cells. The Müller cells are radial glia of the retina and perform a variety of essential functions to maintain retinal homeostasis and respond to stress, injury, or disease. The silent/accessible genes in Müller glia are enriched in pathways related to inflammation, angiogenesis, and other types of cell-cell signaling and were rapidly activated when we tested 15 different physiologically relevant conditions to mimic retinal stress, injury, or disease in human and murine retinae. We refer to these as “pliancy genes” because they allow the Müller glia to rapidly change their gene expression and cellular state in response to different types of retinal insults. The Müller glial cell pliancy program is established during development, and we demonstrate that pliancy genes are necessary and sufficient for regulating inflammation in the murine retina in vivo. In zebrafish, Müller glia can de-differentiate and form retinal progenitor cells that replace lost neurons. The pro-inflammatory pliancy gene cascade is not activated in zebrafish Müller glia following injury, and we propose a model in which species-specific pliancy programs underly the differential response to retinal damage in species that can regenerate retinal neurons (zebrafish) versus those that cannot (humans and mice).

Project description:Purpose: Müller glia are the only glial cell type produced by the neuroepithelial progenitor cells which generate the vertebrate retina. Müller glia are required to maintain retinal homeostasis and support the survival of retinal neurons. Furthermore, they function as an adult stem cell, mediating retinal regeneration among select vertebrate classes. However, the mechanisms which regulate Müller development are poorly understood as considerable overlap exists in gene expression between retinal progenitor cells and differentiated Müller glia. We investigate the functional role of the LIM homeodomain transcription factor Lhx2 in the specification and development of Müller glia in the mouse. Methods: RNA-Seq was performed in collaboration with the Johns Hopkins School of Medicine Deep Sequencing and Microarray Core Facility. Libraries were prepared using Illumina TruSeq RNA Sample kit (Illumina, San Diego, CA) following manufacturer’s recommended procedure. The PCR amplified library was purified using RNAClean XP magnetic beads (Agencourt, Beverley, MA) and run out on a High Sensitivity DNA Chip (Agilent, Santa Clara, CA) for quality check. We used STAR to align RNA-Seq reads onto Ensembl mouse genome GRCm38, release 72. To generate the stand attribute for alignments containing splice junctions, we used the outSAMstrandField intronMotif program. The spliced alignments without strand definition were removed. Number of reads mapped to exons was counted by htseq-count. Genes expressed at very low levels were omitted from further analysis. Gene expression differences between wildtype and mutant samples, significance (p-value) and false discovery rate (FDR) were computed using the generalized linear models based EdgeR. Results: We observed a substantial reduction in expression of Notch pathway genes including Notch1, the Notch ligands Dll1 and Dll3, as well as gliogenic Notch effector genes such as Hes1, Hes5, Id1 and Sox8 and the Müller-gliogenic factor Rax. We likewise observe a substantial reduction in expression of progenitor-specific genes such as Vsx2 and Fgf15. Furthermore, we observed a decrease in the expression of early-onset glial markers such as Crym , Spon1, and Car2.

Dataset Information

Mus musculus

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets