Dataset Information

Proteomic Analysis of Retinal Mitochondria-associated ER Membranes Identified Novel Proteins of Retinal Degeneration in Long-term Diabetes

ABSTRACT: The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is the physical contact site between the ER and the mitochondria and plays a vital role in the regulation of calcium trafficking and bioenergetics. Previous studies suggest that disturbances in calcium trafficking and dysregulation of reactive oxygen species in the ER and mitochondria may contribute to the pathogenesis of diabetic retinopathy (DR). However, few studies have examined the impact of diabetes on the retinal MAM proteome profile. In the present study, we used Long Evans rats with streptozotocin-induced long-term Type 1 diabetes to identify key pathways and proteins in the retinal MAM that are potentially implicated in the pathogenesis of DR.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Retina

DISEASE(S): Diabetes Mellitus

SUBMITTER: Shichen Shen

LAB HEAD: Jun Qu

PROVIDER: PXD033040 | Pride | 2022-10-15

REPOSITORIES: Pride

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			Action	DRS
	B03_02_160826_MAM_new_NDM_1_HCD_OT_4ul_PL.mzid.gz	Mzid
	B03_02_160826_MAM_new_NDM_1_HCD_OT_4ul_PL.raw	Raw
	B03_02_160826_MAM_new_NDM_1_HCD_OT_4ul_PL.raw.mzXML	Mzxml
	B03_03_160826_MAM_new_DM_1_HCD_OT_4ul_PL.mzid.gz	Mzid
	B03_03_160826_MAM_new_DM_1_HCD_OT_4ul_PL.raw	Raw

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Publications

Proteomic Analysis of Retinal Mitochondria-Associated ER Membranes Identified Novel Proteins of Retinal Degeneration in Long-Term Diabetes.

Wang Joshua J JJ Park Karen Sophia KS Dhimal Narayan N Shen Shichen S Tang Xixiang X Qu Jun J Zhang Sarah X SX

Cells 20220909 18

The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is the physical contact site between the ER and the mitochondria and plays a vital role in the regulation of calcium signaling, bioenergetics, and inflammation. Disturbances in these processes and dysregulation of the ER and mitochondrial homeostasis contribute to the pathogenesis of diabetic retinopathy (DR). However, few studies have examined the impact of diabetes on the retinal MAM and its implication in DR pathogenesis. I ...[more]

PMID: 36139394

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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. 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.

Project description:The aim of the study was to explore the effect of topically administered sitagliptin, an inhibitor of the enzyme dipeptidyl peptidase-4, on the retinal expression patterns of an experimental model of DR. Background: Synaptic dysfunction and neuronal damage have been extensively associated with diabetic retinopathy (DR). Our group evidenced that chronic hyperglycemia reduces the retinal expression of presynaptic proteins, which are crucial for a proper synaptic function. The aim of the study was to explore the effect of topically administered sitagliptin, an inhibitor of the en-zyme dipeptidyl peptidase-4, on the retinal expression patterns of an experimental model of DR. Methods: Transcriptome analysis was performed comparing the retinas of 10 diabetic (db/db) mice randomly treated with sitagliptin eye drops (10 mg/mL) twice daily, and the reti-nas of 10 additional db/db mice that received vehicle eye drops. 10 non diabetic mice (db/+) were used as control group. Gene Ontology (GO) and Reactome databases were used to assess the Gene Set Enrichment Analysis (GSEA) in order to explore the most enriched biological pathways among groups. The most differentiated genes of these pathways were validated through quantitative RT-PCR. Results: Transcriptome analysis revealed that sitagliptin eye drops have a significant effect on retinal expression patterns and that neurotransmission was the most enriched biological process. Our study evidenced enriched pathways that contain genes involved in membrane trafficking, transmission across chemical synapses, vesi-cle-mediated transport, neurotransmitter receptors and postsynaptic signal transmission with negative regulation of signaling as a consequence of neuroprotector treatment with sitagliptin. This improves the modulation of macromolecule biosynthetic process with positive regulation of cell communication which provides beneficial effects for neuronal metabolism. Conclu-sions: This study suggests that topical administration of sitagliptin ameliorates the abnormali-ties on presynaptic and postsynaptic signal transmission during experimental DR, and that this improvement is one of the main mechanisms behind the previously demonstrated beneficial effects.