Dataset Information

Quantitative proteomic analysis after neuroprotective MyD88 inhibition in the retinal degeneration 10 mouse

ABSTRACT: Progressive dysfunction and death of photoreceptors occurs in blinding diseases such as age-related macular degeneration and retinitis pigmentosa. The MyD88 protein is a central adaptor molecule for innate immune system Toll-like receptors (TLR) and interleukin-1 receptor (IL-1R), which are active in retinal disease and induce cytokine secretion from inflammatory cells. We recently demonstrated that inhibiting MyD88 in mouse models of retinal degeneration led to increased rod photoreceptor survival, which was associated with altered cytokine expression and increased neuroprotective microglia. However, additional molecular changes associated with MyD88 inhibitor-mediated neuroprotection are not known. Quantitative proteomics using iTRAQ LC-MS/MS is a high-throughput method ideal for providing new information about the molecular mechanisms contributing to photoreceptor protection. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) labeling followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) for quantitative proteomic analysis on the rd10 mouse model of retinal degeneration to identify protein pathways changed by MyD88 inhibition. A total of 42 proteins were differentially expressed in retinas from mice treated with MyD88 inhibitor compared with control. Notably, increased expression of multiple crystallins and chaperones that respond to cellular stress and have anti-apoptotic properties were identified in the MyD88 inhibited mice. Additional differentially expressed biological processes included pyrophosphatase activity and peptide biosynthesis. These data suggest that inhibiting MyD88 may enhance chaperone-mediated tissue protective pathways. Therefore, this study provides new insight into molecular events that contribute to photoreceptor protection from modulating inflammation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Retina

SUBMITTER: Sanjoy Bhattacharya

LAB HEAD: Sanjoy K. Bhattacharya

PROVIDER: PXD024501 | Pride | 2022-02-17

REPOSITORIES: Pride

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			Action	DRS
	Hackam_ITRAQ.mzML	Mzml
	Hackam_ITRAQ.mzid.gz	Mzid
	Hackam_ITRAQ.raw	Raw

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Quantitative proteomic analysis after neuroprotective MyD88 inhibition in the retinal degeneration 10 mouse.

Carmy-Bennun Tal T Myer Ciara C Bhattacharya Sanjoy K SK Hackam Abigail S AS

Journal of cellular and molecular medicine 20210925 20

Progressive photoreceptor death occurs in blinding diseases such as retinitis pigmentosa. Myeloid differentiation primary response protein 88 (MyD88) is a central adaptor protein for innate immune system Toll-like receptors (TLR) and induces cytokine secretion during retinal disease. We recently demonstrated that inhibiting MyD88 in mouse models of retinal degeneration led to increased photoreceptor survival, which was associated with altered cytokines and increased neuroprotective microglia. Ho ...[more]

PMID: 34562309

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Project description:Retinal degeneration is the leading cause of irreversible blindness. Retinitis pigmentosa (RP) is a genetically heterogenous group of diseases. In the United States, approximately one in 4000 individuals is affected. RP begins with the loss of night vision due to the loss of rod photoreceptor cells. The disease progresses slowly with the loss of peripheral vision, and eventually leads to complete debilitating and irreversible blindness. The first mutation associated with human RP was identified in the gene encoding rhodopsin, the G-protein coupled receptor of rod photoreceptor cells. Mutations within the rhodopsin gene account for significant portion of RP cases. Specifically, mutations of the proline at residue 347 in rhodopsin have been linked to human RP. We are fortunate to have access to the P347S rhodopsin mutant mice. These mice represent an excellent transgenic mouse model of retinal degeneration. The P347S rhodopsin mutation is one of the best studied mutations, yet the mechanism by which the mutation causes degeneration is still unknown. One study has demonstrated that galectin-1 plays a role in degeneration of neuronal processes (1) and another study has shown that expression level of galectin-3 is elevated in retinas of patients with age-related macular degeneration. These studies in conjunction with the availibility of the P347S mutant mice have provided impetus to examine the pathogenesis of retinal degeneration in the context of the possible role of glycans and glycan-binding proteins. The time course of photoreceptor degeneration in the P347S mouse model has been carefully studied. In these mice, degeneration is barely detectable at 1 month of age, yet biochemical evidence suggests that the rod photoreceptor cells have already begun to die. At 4 months of age, approximately half of the rod photoreceptor cells have degenerated. To distinguish involvement of glycogens at the various stages of retinal degeneration, we have collected retinas of wild type and the mutant mice at four time points (1, 2, 3, and 4 months of age). This will allow us to identify the genes that target early, mid- and late stages of the retinal degeneration process. Thus we request the analysis of total 24 samples as specified below: Age Group (months) Mice No of samples at each time point 1 Wild type 3 2 Wild type 3 3 Wild type 3 4 Wild type 3 1 P347S 3 2 P347S 3 3 P347S 3 4 P347S 3 Total 24.

Project description:Photoreceptor disorders are collectively known as retinal degeneration (RD), and include retinitis pigmentosa (RP), cone-rod dystrophy and age related macular degeneration (AMD). These disorders are largely genetic in origin; individual mutations in any one of >200 genes cause RD, making mutation specific therapies prohibitively expensive. A better treatment plan, particularly for late stage disease, may involve stem cell transplants into the photoreceptor or ganglion cell layers of the retina. Stem cells from young mouse retinas can be transplanted, and can form photoreceptors in adult retinas. These cells can be grown in tissue culture, but can no longer form photoreceptors. We have used microarrays to investigate differences in gene expression between cultured retinal progenitor cells (RPCs) that have lost photoreceptor potential, postnatal day 1 (pn1) retinas and the postnatal day 5 (pn5) retinas that contain transplantable photoreceptors. We have also compared FACS sorted Rho-eGFP expressing rod photoreceptors from pn5 retinas with Rho-eGFP negative cells from the same retinas. We have identified over 300 genes upregulated in rod photoreceptor development in multiple comparisons, 37 of which have been previously identified as causative of retinal disease when mutated. It is anticipated that this research should bring us closer to growing photoreceptors in culture and therefore better treatments for RD. This dataset is also a resource for those seeking to identify novel retinopathy genes in RD patients. We extracted whole retinas from postnatal day 1 (Pn1) and postnatal day 5 (Pn5) mice, and compared them with cultured RPCs derived from pn5 retinas, using Affymetrix mouse 430A_2 arrays. We also extracted cells from Rho-eGFP Pn5 retinas and FACS sorted them. GFP+ve cells represent immature rod photoreceptors, as they express the Rho-eGFP fusion protein, which is only expressed in rods. GFP-ve cells represent all other retinal neurons. These samples were amplified and compared using Affymetrix mouse 430A_2arrays, by Source Biosciences GMBH, Berlin, Germany. Results from immature rods were then compared with those from other retinal neurons, while results from whole Pn5 retinas were compared with Pn1 retinas (which don't yet express rod specific genes), and RPCs, which are glial precursors. RPCs were also compared with Pn1 retinas. Genes which showed changed expression profiles in at least 3/4 of comparisons were prioritised for further investigation.