Dataset Information

Multi-omics profiling of Müller cells in diabetic retinopathy identifies the glucocorticoid receptor as a master regulator of gliotic response

ABSTRACT: Diabetic retinopathy (DR), the leading cause of blindness in working-age adults, is thought to be primarily a microvascular complication of diabetes. As a central element of the retinal neurovascular unit, Müller cells, the major macroglia of the retina, are important for maintaining a healthy and functional retina and play a critical role in several pathological events during DR disease progression. Here, we aim to improve our understanding of Müller cell-specific signalling pathways that are altered during DR disease progression in order to develop novel gene therapy strategies that target Müller cells. We used a multi-omics approach on purified Müller cells from 6-month-old control and diabetic db/db mice, including (i) RNA-seq followed by oPOSSUM-3 transcription factor (TF) binding site cluster analysis, (ii) glial chromatin landscape analysis by ATAC-seq, and (iii) Müller cell proteomics by MS/MS mass spectrometry. This led to the identification of the glucocorticoid receptor (GR, gene ID: Nr3c1) most highly expressed in Müller cells. In cells from diabetic mice, GR mRNA and protein expression is significantly reduced and its target gene cluster downregulated in Müller cells. Importantly, GR was identified as a potential master regulator not only by oPOSSUM analysis based on differentially expressed mRNA in Müller cells, but also validated by the ATAC-seq approach. In an in vitro cortisol-stimulated retinal explant model cortisol not only increased GR phosphorylation, but also induced changes in the expression of known downstream GR target genes. Finally, we evaluated whether AAV-mediated GR overexpression in Müller cells improves retinal functionality and we found moderate improvements indicated by electroretinography. While synthetic and topical glucocorticoids are widely used in ophthalmology with undeniable beneficial effects, our study provides valuable new insight into the role of GR signalling and glial alterations in the diabetic retina. This supports the therapeutic concept of locally enhancing the GR signaling axis. Our findings of reduced GR levels in Müller cells of the diabetic retina suggests that therapeutic approaches should aim at increasing the expression of the receptor rather than adding more ligand.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Retina

SUBMITTER: Stefanie Hauck

LAB HEAD: Antje Grosche

PROVIDER: PXD045085 | Pride | 2024-05-23

REPOSITORIES: Pride

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			Action	DRS
	GRF7778A11MA3-40-288292.raw	Raw
	GRF7778A12MA4-40-288293.raw	Raw
	GRF7778A13MA3-40-288410.raw	Raw
	GRF7778A14MA3-40-288411.raw	Raw
	GRF7778A15MA3-40-288427.raw	Raw

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Publications

The glucocorticoid receptor as a master regulator of the Müller cell response to diabetic conditions in mice.

Pfaller Anna M AM Kaplan Lew L Carido Madalena M Grassmann Felix F Díaz-Lezama Nundehui N Ghaseminejad Farhad F Wunderlich Kirsten A KA Glänzer Sarah S Bludau Oliver O Pannicke Thomas T Weber Bernhard H F BHF Koch Susanne F SF Bonev Boyan B Hauck Stefanie M SM Grosche Antje A

Journal of neuroinflammation 20240125 1

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significa ...[more]

PMID: 38273366

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Project description:Diabetic retinopathy (DR), the leading cause of blindness in working-age adults, is thought to be primarily a microvascular complication of diabetes. Müller cells, the major macroglia of the retina, are important for maintaining a healthy and functional retina and play a critical role in several pathological events during DR disease progression as they are a central element of the retinal neurovascular unit. Here, we aim to improve our understanding of Müller cell-specific signaling pathways that are altered during disease progression in order to develop novel gene therapy strategies targeting Müller cells in DR. To achieve this, we used a multi-omics approach on purified Müller cells from 6-month-old control and diabetic db/db mice, including (i) RNA-seq followed by oPOSSUM-3 transcription factor (TF) binding site cluster analysis, (ii) glial chromatin landscape analysis by ATAC-seq, and (iii) Müller cell proteomics by MS/MS mass spectrometry. This led to the identification of the glucocorticoid receptor (GR, gene ID: Nr3c1) as the most promising candidate. Its transcripts and protein were most highly expressed in Müller cells and significantly reduced in cells from diabetic mice, its target gene cluster was downregulated in Müller cells from diabetic animals, and importantly, it was identified as a potential master regulator not only by oPOSSUM analysis based on differentially expressed mRNA in Müller cells, but also validated by the ATAC-seq approach. Next, we investigated the effect of GR modulation in an in vitro cortisol-stimulated retinal explant model. Cortisol not only increased GR phosphorylation levels, but also induced changes in the expression of known downstream GR target genes. Finally, we evaluated whether AAV-mediated GR overexpression in Müller cells improves retinal functionality and found moderate functional improvements in electroretinogram recordings. Although synthetic and topical glucocorticoids are widely used in ophthalmology with undeniable beneficial effects, our study provides valuable new insights into the role of GR signaling and glial alterations in the diabetic retina and thus supports the therapeutic concept of locally enhancing the GR signaling axis. However, our finding of reduced GR levels in Müller cells of the diabetic retina suggests that therapeutic approaches should aim at increasing the expression of the receptor rather than adding more ligands. This may result in optimized local efficacy with fewer systemic unwarranted side effects.