Aging is a pivotal driving force behind various degenerative processes that culminates in functional impairment and stochastic molecular changes, which predispose to chronic disease conditions. In the eye, progressive deterioration of the ocular neurovascular function is exacerbated by aging, particularly in neurodegenerative diseases such as glaucoma, which is the second cause of vision loss worldwide. To add to the complexity, there is gender predilection that is not clearly demarcated in retinopathies. We recently deciphered the neuro-vasculoprotective roles of bioactive lipids following inhibition of soluble epoxide hydrolase (sEH/ Ephx2) in the CYP pathway in male mice, alluding to a potential druggable target to mitigate the impact of aging on visual dysfunction. This study tested the hypothesis that there are distinct gendered differences in the proteome of aged retina of the Ephx2 gene knockout (KO) mice.

Retinae were isolated from 160 young (3-5 months) and old (12-24 months) wild type (WT) and KO mice of both sexes. Samples were pooled (n=5 mice/group/replicate) with 4 biological replicates per group and subjected to label-free mass spectrometry-based proteomics using a Thermo Q Exactive Plus Hybrid Quadrupole-Orbitrap MS coupled to an EASY-nLC 1000 uHPLC system. The MS data analysis were followed by bioinformatics analysis with Ingenuity Pathway Analysis (IPA) to unravel protein functionalities and corresponding upstream regulators that govern the protein expression.

We identified a total of 3706 retinal proteins with at least 2 unique peptides and FDR < 1 %. A significant (p < 0.05) number of differentially expressed protein clusters in the aging retina were implicated in the eIF2 and sirtuin signaling pathways in both sexes. Intriguingly, proteins related to neurodegenerative processes (p=9.9×10^-4) that cause retinal diseases (p=9.6×10^-4) and oxidative stress (p=2.6×10^-4) were significantly regulated only in aged WT female. However, KO resulted in significant activation of cell-cell contact (p=1.2×10^-3) and neuritogenesis (p=5.4×10^-5) in both genders. The female sex showed striking resilience against ferroptosis, while the male counterparts demonstrated highly significant activation of this signaling pathway in both WT (z-score: 1.3) and KO mice (z-score: 2). Likewise, XBP1, an upstream transcription regulator with ER stress-ameliorating function, was found in only male retinae. Remarkably, this regulator was found to be significantly activated in the WT mice (p=3.9×10^-4, z-score: 0.5) but inhibited in the KO male mice (p=8.1×10^-3, z-score: -1.2).

Taken together, our study characterized for the first time the impact of sex differences in the retinal proteome of KO mice, which is a key determinant in understanding the pathomechanisms underlying age-related retinal diseases. Our findings will be instrumental for development of personalized intervention targeting sEH for retinopathies that present a predilection for a particular sex.