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    (2022) Zaman, Khadiza; Nguyen, Vien; De La Cruz, Daniel; Prokai-Tatrai, Katalin; Prokai, Laszlo
    Purpose: The retina is highly vulnerable to age-associated neurodegeneration critically affecting its nerve cells, which has prompted hitherto mostly futile searches to identify retinal neuroprotectants. Recent proteomics studies have revealed that estrogens elicit a variety of beneficial effects on retinal health in females. Here, we detail our proteomics studies showing the impact of 17β-estradiol (E2) eye drops on male rat retina's with focus on affected protein networks and associated biological processes. Methods: Orchidectomized (ORX) Brown Norway rats received either 0.1% w/v E2 eye drops in saline/2-hydroxypropyl-β-cyclodextrin vehicle or the vehicle only once daily for three weeks. Proteins from target tissues were extracted and analyzed by mass spectrometry-based proteomics using label-free quantification (LFQ). MS/MS data were searched against the UniProt rat protein database by Mascot (Matrix Science). Validations and LFQ to detect statistically significant changes in protein abundances between groups were performed using Scaffold (Proteome Software). Mapping of the differentially expressed proteins to protein interaction networks and biological processes was done through Ingenuity Pathway Analysis® (Qiagen). Results: Our shotgun proteomics relying on LFQ covered 1761 protein, with 139 proteins differentially regulated. With identical treatment regimen and experimental methodology to collect data, the number of E2-regulated proteins in the male rat retina was less than half of what we found in the female rat retina. However, in terms of regulation, like our findings for females, the top network in the male retina was linked to development disorder, ophthalmic disease, organismal injury and abnormalities The top canonical pathways associated with this network was protein ubiquitination and synaptogenesis signaling. Another strong aspect of protein interactions was the involvement of several upregulated isoforms of crystalline driving the top network. The abundant presence of crystallins has been found to promote the survival of retinal ganglion cells upon age-associated stress and traumatic insults, while their suppression is associated with retinal neurodegeneration. Conclusion: Our study captured E2's beneficial effects on the male rat retina linked to regulation of various neuroprotective pathways like estrogen-receptor signaling, synaptogenesis stimulating efficient protein disposal, and mitochondrial respiratory chain biogenesis to maintain retinal health. Targeted proteomics are in progress to validate a subset of E2-regulated proteins as robust target engagement markers for preclinical studies aimed at assisting the development of the hormone's retina-selective delivery to assure its therapeutic safety in males after topical treatment.