Abstract
Purpose :
A variety of conditioning treatments share a strong preclinical track record for epigenetically modifying the retina in ways that protect it from injury and disease [1]. Translational relevance is enhanced by the demonstration that repetitive conditioning can extend the disease-resilient phenotype long after treatment [2-4]. In the present study, we performed quantitative mass spectrometry (MS) on retinae from male and female mice following 2 wks of repetitive hypoxic conditioning (RHC) to gain a proteome-wide understanding of the epigenetically modified phenotype induced prior to injury.
Methods :
Adult, outbred Swiss-Webster ND4 mice of both sexes were treated with RHC, or served as normoxic controls; whole retinae (without choroid/RPE) were harvested the following week and processed for MS3 analysis on a Fusion Orbitrap MS using TMTpro labels for quantification, with <5% FDR. Differentially expressed proteins (>1.1 fold-change) were analyzed bioinformatically (z-score>2) (IPA, Qiagen).
Results :
MS identified and quantified 6253 proteins, 688 of which were differentially expressed in response to RHC across both sexes. The expression of 166 of these proteins changed uniquely in males or females, whereas that of 522 proteins (216 upregulated and 306 downregulated) were sex-independent. Findings in the latter group implicated increased nuclear (MAF, SOX17, TP53, WT1) and mitochondrial (TFB1M, PHB) transcription, enhanced blood-retinal barrier integrity (PTGDS, MAPK7), increased ceramide synthesis (SMPD1), and Akt/mTOR activation (PIP4P1, KRT17). Bioinformatic analyses of these shared proteins predicted RHC-induced changes in the expression of proteins involved in diverse signaling and metabolic pathways consistent with the promotion of cell survival/anti-apoptotic signaling and free radical scavenging, and a reduction in inflammation, microglial reactivity, microtubule dynamics, and cell migration/movement.
Conclusions :
RHC induces widespread and sustained changes in the retinal proteome prior to injury/disease. This 'prime-the-pump' phenotype, conserved across sexes, is foundational to understanding innate, epigenetically-induced mechanisms of neuroprotection that could be leveraged for therapeutics.
References: [1] Gidday JM, Cond Med 2018; [2] Zhu Y et al., IOVS 2007; [3] Zhu Y et al., Mol Med 2012; [4] Harman JC et al., IOVS 2020.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.