Abstract
Purpose :
MyD88 mediates inflammatory signaling in the retina. MyD88 inhibition delayed rod photoreceptor death in two mouse models of retinal degeneration. In this study, we performed quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry iTRAQ to characterize early protein changes that may contribute to the protective effects of inhibiting MyD88 in rd10 mouse retinas.
Methods :
Rd10 mice (male and female, PN day 18) were injected IP with 2 mg/Kg MyD88 inhibitor (neuroprotective dose) or control peptide (n=4 each). Retinas were collected 3 days later, proteins were extracted then trypsin digested, and incubated with 8-PLEX iTRAQ Reagents followed by analysis using Q Exactive mass spectrometer. Proteins were identified and quantified using Proteome Discoverer 2.2 software, bioinformatics enrichment analysis used PANTHER v.15.0 and interaction networks used the STRING database v.11. QPCR was used to confirm gene expression.
Results :
A total of 332 proteins were identified with high (FDR<0.01) and medium confidence (FDR<0.05) with >2 unique matching peptides. Forty-two proteins were differentially expressed (fold change >1.2 or <0.83; p<0.05): 20 proteins were upregulated in the MyD88 inhibitor group and 22 were downregulated. The top biological processes were metabolic, developmental and regulation, and the top molecular functions were catalytic activity, binding and structural molecules. The upregulated proteins were enriched in crystallins, unfolded protein binding and structural molecular activity, and decreased proteins were enriched in pyrophosphatase activity, small molecule binding, and peptide biosynthesis. Notably, MyD88 inhibition led to upregulation of 7 stress-response crystallins. STRING analysis on the upregulated proteins showed 3 distinct physical interaction networks (p=1.94e-05): crystallins, cytosolic small ribosome subunit and aminopeptidase activity. Retinal expression of the crystallin genes was confirmed by QPCR.
Conclusions :
This study provides a foundation for understanding molecular mechanisms regulating retinal homeostasis in rd10 mice. A novel link was identified between anti-apoptotic crystallins and MyD88 inflammatory pathway inhibition.
This is a 2021 ARVO Annual Meeting abstract.