Browsing by Author "Zaman, Khadiza"
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Item A DISCOVERY-DRIVEN LABEL-FREE PROTEOMICS BASED SURVEY OF ESTRADIOL-REGULATED PROTEIN NETWORKS AND ASSOCIATED BIOLOGICAL FUNCTION IN THE RAT RETINA(2019-03-05) Rahlouni, Fatima; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo; Zaman, KhadizaA DISCOVERY-DRIVEN LABEL-FREE PROTEOMICS BASED SURVEY OF ESTRADIOL-REGULATED PROTEIN NETWORKS AND ASSOCIATED BIOLOGICAL FUNCTION IN THE RAT RETINA Khadiza Zaman1, Fatima Rahlouni1,2, Vien Nguyen1, Katalin Prokai-Tatrai1 and Laszlo Prokai1 1 Department of Pharmacology & Neuroscience,University of North Texas Health Science Center, Fort Worth, TX. 2 Texas College of Osteopathic Medicine, University of North Texas Health Science Center, Fort Worth, TX. Purpose: Previous studies have established the pleiotropic role of 17β-estradiol (the predominant human estrogen) as a potent neuroprotectant, but only recently it has gained attention for its therapeutic potential against ocular neurodegenerative diseases. Thus, this study was designed to perform a label free quantitative proteomics based survey to understand the impact of topical administration of E2 on the rat retina. This is one of the first reports elucidating E2-regulation of rat retinal proteins, networks and associated biological processes, thus providing us with more insights on topical hormone therapy. Methods: Ovariectomized (OVX) Brown Norway rats were given 0.1% w/v E2 eye drops in saline/2-hydroxypropyl-β-cyclodextrin vehicle and controls received vehicle daily for three weeks. Retina from euthanized animals were immediately isolated. Retinal proteins were extracted and analyzed using data-dependent nanoflow LC-ESI-MS/MS on Orbitrap EliteTM (Thermo) or Orbitrap Velos Pro. MS/MS data was searched against the UniProt rat protein database using Mascot (Matrix Science). Validations and label-free quantitation were performed using Scaffold (Proteome Software) by observing changes in protein abundances between treated and control using t test. Differentially expressed proteins were mapped to protein interaction networks and biological processes through Ingenuity Pathway Analysis® (Qiagen). Results: In our proteomics-based quantitation, we identified 66 E2 regulated proteins in the OVX rat retina among which 49 up-regulated and 17 down-regulated (p1.5-fold change between groups). Some of the most highly scored identified networks are associated with endocrine system disorders, organismal injury and abnormalities, and developmental disorder. Presence of nuclear estrogen receptor (ER) in our dataset also reinforces the intricate nature of E2 signaling conveying neuroprotection. Our network-based analysis emphasized on the role of E2 in neuroprotection through regulation of various stress-induced signaling cascades such as ERK/MAPK pathways. Conclusion: By using an OVX model with little or no endogenous E2, our study potentiates the neuroprotective role of E2 upon topical administration of the hormone. With this vast array of information on estrogen biology we seek to create foundations in basic science research regarding hormone therapy focusing on the “estrogenic retina.” Acknowledgement: This study was supported by the National Eye Institute and the Office of Research on Women’s Health (grant number EY027005 to K.P.-T.) and by the Robert A. Welch Foundation (endowment BK-0031 to L.P.).Item Comparative Proteomics Analysis Reveals Unique Early Signaling Response of Saccharomyces cerevisiae to Oxidants with Different Mechanism of Action(MDPI, 2020-12-26) Pandey, Prajita; Zaman, Khadiza; Prokai, Laszlo; Shulaev, VladimirThe early signaling events involved in oxidant recognition and triggering of oxidant-specific defense mechanisms to counteract oxidative stress still remain largely elusive. Our discovery driven comparative proteomics analysis revealed unique early signaling response of the yeast Saccharomyces cerevisiae on the proteome level to oxidants with a different mechanism of action as early as 3 min after treatment with four oxidants, namely H2O2, cumene hydroperoxide (CHP), and menadione and diamide, when protein abundances were compared using label-free quantification relying on a high-resolution mass analyzer (Orbitrap). We identified significant regulation of 196 proteins in response to H2O2, 569 proteins in response to CHP, 369 proteins in response to menadione and 207 proteins in response to diamide. Only 17 proteins were common across all treatments, but several more proteins were shared between two or three oxidants. Pathway analyses revealed that each oxidant triggered a unique signaling mechanism associated with cell survival and repair. Signaling pathways mostly regulated by oxidants were Ran, TOR, Rho, and eIF2. Furthermore, each oxidant regulated these pathways in a unique way indicating specificity of response to oxidants having different modes of action. We hypothesize that interplay of these signaling pathways may be important in recognizing different oxidants to trigger different downstream MAPK signaling cascades and to induce specific responses.Item Discovery-driven Label-free Quantitative Proteomics Study to Understand Estradiol-mediated Neuronal Processes in the Hippocampus and its Implication in Alzheimer’s Disease in Ovariectomized Rats(2024-03-21) Neagu, George; Zaman, Khadiza; Nguyen, Vien; Kapic, Ammar; Prokai-Tatrai, Katalin; Prokai, LaszloPurpose: Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder that affects millions of people globally. Studies report an increased susceptibility to the development of AD in post-menopausal women. There is renewed interest in utilizing estrogen therapies due to its neuroprotective effects on the brain; however, the mechanisms of these neuroprotective effects are poorly understood. The hippocampus is involved in memory formation and is a critical region where early damage in AD is often seen. This discovery-driven proteomics study elucidates several candidate proteins and biological pathways mediated via E2 and implicated in neurological signaling in the hippocampus. Methods: Ovariectomized female Sprague-Dawley rats were treated with daily subcutaneous injections of either vehicle or 50μg/kg E2 for five days before the rats were sacrificed with the hippocampus collected for proteomics. Protein extracts were taken from centrifugated hippocampal tissue and prepared via a series of steps including urea incubation, disulfide bond reduction, carbamidomethylation of thiol groups, and digestion via trypsin with subsequent quenching. The digested proteins were dried, reconstituted in solvent, and processed via nano-LC-MS/MS. The MS/MS spectra were searched against a Rattus norvegicus proteome database for peptide fragment and protein identification via ProteomeDiscover (Thermo Fisher Scientific) using Mascot as a search engine and validated using Scaffold (Proteome Software). Bioinformatic analysis using Ingenuity Pathway Analysis (Quiagen) allowed the construction of associative and predicted protein networks. Results: The processed MS/MS data proteins revealed several candidates for future targeted validation. Among those proteins, calcium/calmodulin-protein kinase II implicated in memory and learning processes, such as long-term signal potentiation in the hippocampus, was more abundant in the treatment group (p = 0.00052) with fold changes in the protein cluster abundances ranging from 1.3 to 1.5 versus control. Other proteins, such as microtubule-associated protein tau, implicated in AD, also had a marked fold change of –2.5 abundance in treatment versus control (p = 0.00014). There substantial overall difference in protein abundances for neurological disease pathways, including AD, identified in Ingenuity Pathway Analysis (p = 8.9e-7) as well as in pathways involving nervous system development and function (p= 4.57e-7) between E2-treated and vehicle-treated rats. Conclusion: This dataset analysis aims to evaluate the effects of E2 on the proteome of the hippocampus in ovariectomized rats. The evident increased fold changes in calcium-dependent and calcium-associated proteins in the context of neuronal processes suggest increased downstream modulation of synaptic signaling, which could be further examined by microdialysis assay of neurotransmitters. Future studies utilizing microdialysis may examine E2’s estrogen receptor-mediated effects on cholinergic neuronal signaling in the hippocampus, which could further understand AD in the context of cholinergic neuron loss.Item Identification of proteins affected by increased intraocular pressure in the glaucomatous female mouse retina by label-free proteomics(2023) Zaman, Khadiza; Morgan, Autumn B.; Nguyen, Vien; Prokai-Tatrai, Katalin; Inman, Denise; Prokai, LaszloPurpose: Mass spectrometry-based retina proteomics using animal models of human diseases has enabled novel insights into ocular neuropathology’s such as in glaucoma, as it holds promise for disease biomarker discovery. However, publicly accessible data on retina proteins affected by ocular hypertension (OHT) in animal models utilized males, or sex was not disclosed. Recently, female animals were chosen to advance therapeutic antibody development against glaucomatous neurodegeneration with retina proteomics support. Therefore, our retinal proteomics-based investigation intended to fill a knowledge gap by focusing on OHT-induced changes of protein expressions in the glaucomatous female retinae compared to normotensive controls. Methods: Proteins were extracted from the retinae of normotensive female mice (control, n=5) and OHT mice (n=5) in which increase of intraocular pressure was induced by the magnetic microbead method. After reduction, alkylation and digestion by trypsin, bottom-up shotgun proteomics analyses of the samples were done using data-dependent nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) on a hybrid Orbitrap instrument (Thermo Fisher Scientific). MS/MS spectra were searched against the UniProt mouse protein sequence database using the SEQUEST search engine in Proteome Discoverer (version 2.4; Thermo Fisher Scientific). Validation of proteins identifications using stringent criteria and label-free quantifications (LFQ) employing spectral counting to detect regulated proteins between groups using t-tests were performed using Scaffold (version 5.1.2; Proteome Software). Targeted proteomics on selected biomarkers was designed and analyzed using SkylineTM (MacCoss Lab software). Mapping to protein interaction networks and biological processes was done through Ingenuity Pathway Analysis® (IPA®, Qiagen). Results: Our discovery driven data-dependent nanoflow LC–ESI-MS/MS analyses covered nearly 1200 retinal proteins with <1% false discovery rate. Among these proteins, 168 were significantly affected by OHT based on LFQ. Bioinformatics analyses by IPA® revealed important diseases and functions triggered by OHT pertaining to neurological and ophthalmic pathologies. The topmost protein interaction network represented neurological disease, organismal injury and abnormalities. The molecule activity predictor of IPA® revealed important canonical pathways, including inhibition of synaptogenesis signaling and mitochondrial dysfunction leading to degeneration of central nervous system tissue. Another prominent protein interaction network represented nervous system development and function, as well as organ development. In addition, this network also displayed downregulation of neuroprotective crystallins owing to OHT. Neuronal crystallins have been identified not only as biomarkers to monitor the progression of OHT-induced retinal neuropathy and evaluate neuroprotective interventions, but also as potential druggable targets or possible protein therapeutics to prevent glaucomatous neurodegeneration. Parallel reaction monitoring-based targeted proteomics validation of significant OHT-regulated retina proteins are currently underway to establish them as potential preclinical biomarkers and/or therapeutic targets. In addition, our studies will be expanded to investigate sex as a biological variable affecting ocular neurodegeneration associated with glaucoma. Conclusion: We anticipate that biological information one can derive from our dataset at the protein expression level will provide inspiration for future hypothesis-driven experimental studies focusing on knowledge gaps involving the biology of glaucomatous neurodegeneration.Item LABEL-FREE QUANTITATIVE PROTEOMICS REVEAL PROTEIN NETWORKS AND ASSOCIATED BIOLOGICAL PROCESSES IN ANDROGEN DEPRIVED MOUSE SEMINAL VESICLES IN RESPONSE TO 17-β-ESTRADIOL (E2) TREATMENT(2023) Kapic, Ammar; Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloPurpose: Androgen deprivation therapy (ADT) remains the primary treatment strategy for inhibiting prostate cancer progression. However, systemic ablation of androgen-mediated signaling induces various metabolic disorders, cognitive decline, and osteoporosis. Therefore, like in menopausal women, 17-β-estradiol (E2) supplementation has been suggested as a treatment to reduce side effects associated with ADT. A recent clinical trial utilizing transdermal estrogen patches reported reduced osteoporosis markers and hot flashes. Estrogen receptors ER) are expressed in the male reproductive system and play a role alongside androgens in maintaining function and growth. Under normal physiological conditions, increased E2 concentrations induce an inhibitory effect on the size of the male reproductive organs, including the seminal vesicles (SV); however, under androgen depletion, E2 supplementation has been reported to reduce the atrophy of the SV in mice. In this study, we report for the first time a discovery-driven proteomic analysis of E2’s effects on the SV in mice under the conditions of surgical castration to model patients undergoing ADT. Methods: Surgically castrated mice (n=4) were subcutaneously injected with E2 (treated group) or vehicle (control) daily for five days and sacrificed to obtain SV. Proteins were extracted, reduced, alkylated, and digested with trypsin for analyses using data-dependent microflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) on LTQ Orbitrap Velos ProTM (Thermo Fisher Scientific). MS/MS data was searched against the UniProt Mouse protein database using Sequest in Proteome Discoverer (Thermo Fisher Scientific) and MaxQuant (Max Planck Institute). Validation of protein and label-free quantification (LFQ), combining spectral counting and total TIC, were performed using Scaffold (Proteome Software) to identify significantly affected proteins. Post-hoc t-test was performed to identify differences in protein abundances between groups. Regulated proteins we mapped to protein interaction networks and biological functions employing Ingenuity Pathway Analysis® (IPA®, Qiagen). Targeted proteomics- sed validation of significant candidate proteins is ongoing, and data will be analyzed using Skyline TM (MacCoss Lab, University of Washington). Results: Our discovery-driven LC–ESI-MS/MS analyses identified 7000 proteins with high confidence from the SV of E2-treated and control mice. IPA®-based bioinformatics of the E2-regulated proteins showed molecular and cellular functions-associated enrichment of carbohydrate metabolism, DNA replication, recombination, and repair, as well as free radical scavenging. The topmost regulated protein interaction network represented cell cycle, cell signaling, and small molecule biochemistry. Enhanced activation of the estrogen receptor β (ESR2) was implicated by the molecule activity predictor (MAP) tool of IPA®. Additionally, MAP predicted that the protein interaction represented within this network might impact disease and physiological processes associated with the proliferation of prostate cancer and regulation of gonadal cells. Furthermore, we were able to screen several preclinical biomarkers that participate in androgen receptor activity, modulating ER-mediated transcription and reproductive system development and function. Targeted proteomics-based validation of these biomarkers is ongoing. Conclusion: Our study aims to provide an in-depth account of the alterations occurring at the protein level in the SVs in response to E2 supplementation during ADT and to select and validate preclinical biomarkers for prognostic and therapeutic applications.Item Metabolic changes in optic nerve head astrocytes following glaucoma-associated deformation(2021) Pappenhagen, Nathaniel; Zaman, Khadiza; Prokai, Laszlo; Inman, DenisePurpose: The astrocytes of the optic nerve head (ONHAs) are the predominant glial cell in the unmyelinated portion of the optic nerve. We tested the metabolic changes these cells undergo in glaucoma by exposing them in vitro to degrees of deformation similarly experienced as a result of increased intraocular pressure in primary open angle glaucoma. Methods: Primary astrocytes were cultured from the cortices of P1 mouse pups or P7 optic nerve head explants then seeded on collagen-coated FlexCell plates. The astrocytes were then biaxially stretched by 12% for 24 hours using the FX-6000T FlexCell. ONHA extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Seahorse XFe24 Analyzer, while changes in the proteome were measured using mass spectrometry. Results: Stretched cortical astrocytes showed at least twofold increases in metabolic proteins such as glutamate dehydrogenase 1, isocitrate dehydrogenase 1, and aldolase fructose-bisphosphate c. There was also at least a twofold decrease in citrate synthase, isocitrate dehydrogenase 2, glycogen phosphorylase B, and adenylate kinase 1. In the Seahorse Analyzer, stretched ONHAs showed increased glycolytic ECAR, maximal ECAR, and maximal OCR. Stretched astrocytes showed no difference in their dependence on pyruvate compared to controls, but a significant decrease in their capacity for mitochondrial respiration from pyruvate. Conclusions: Exposing astrocytes to glaucoma-associated deformation altered their metabolism in ways that indicated stronger commitment to glycolysis compared to control astrocytes, such as increases in glycolytic proteins, decreases in mitochondrial proteins, and increases in glycolytic ECAR.Item Neuroprotective Effects of DHED Eyedrops Protects Visual Function Despite Elevated IOP in an Ocular Hypertension Animal Model(2024-03-21) Kapic, Ammar; Neagu, George; Nguyen, Vien; Zaman, Khadiza; Prokai, Laszlo; Prokai-Tatrai, KatalinPurpose: Glaucoma remains the second leading cause of irreversible blindness and is often associated with chronically elevated intraocular pressure (IOP) leading to ocular hypertension (OHT). All of the currently accepted therapies attempt to reduce the elevated IOP. However, despite intervention, studies show progressive neuronal damage continues in the retina and may extend to the rest of the visual system, leading to additional neuropathologies. Interest in utilizing 17β-estradiol (E2) for its neuroprotective effects has become increasingly recognized; however, due to its side effects, such as cancer risk and feminization in males, its application as a therapy is limited. Our lab has developed the estrogen prodrug, 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED), which remains inactive until its CNS-specific metabolism via short-chain reductase into the active compound E2. This study aims to elucidate the pleiotropic effects of E2 derived from DHED as a potential therapy for preserving the visual system under OHT. We hypothesize that topical application of DHED will prevent the neurodegenerative effects of chronic OHT on the retina and maintain visual function. Methods: OHT was induced in 8 to 10-month-old male Brown Norway rats via hypertonic saline injection into an episcleral vein. IOP was measured via a tonometer (Tonolab) to confirm sustained elevated IOP post-surgery and throughout the treatment period. DHED was topically delivered through eyedrops (20% 2-hydroxylpropyl-beta-cyclodextrin, 0.1% DHED, and saline) once per day. Visual acuity (VA) and contrast sensitivity (CS) were measured using the OptoMotry system with the OptoMotry 1.7 software (Cerebral Mechanics Inc). VA and CS were assessed using the "Rat" preset, and gratings were adjusted using a simple staircase progression. A fixed frequency of 0.272 c/D was chosen for the CS based on prior studies. Observers for the OptoMotry tests were blinded. The eyes and optic nerves were collected and fixed for RGC and axon counts, respectively. Seminal vesicles were collected and weighed to assess peripheral estrogenic effects. Results: The IOP was elevated by 53 % ± 15% and was sustained in both vehicle and DHED-treated groups with no differences between treatment groups. The vehicle-treated group gradually lost visual function, retaining only 60% ± 5% and 30% ± 4% of their VA and CS, respectively, by the end of the treatment period. However, the DHED-treated group maintained significantly better visual performance, retaining 91% ± 3% and 75% ± 7% of their VA and CS compared to the baseline. No differences in the mass of the seminal vesicles between treatment groups. Comparison of RGC and axon counts in the optic nerve are ongoing. Conclusion: This study demonstrates the neuroprotective effects of DHED-derived E2 on the visual system without peripheral side effects. Despite sustained OHT, the VA and CS of the topically administered DHED reduced the impact of injury compared to the vehicle control group. Future studies will investigate DHED administration's impact on the retina and visual cortex proteome.Item PROGRESSING THYROTROPIN-RELEASING HORMONE TOWARDS NEUROTHERAPEUTIC APPLICATIONS(2022) De La Cruz, Daniel; Nguyen, Vien; Zaman, Khadiza; Prokai, Laszlo; Prokai-Tatrai, KatalinPurpose: Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) is a small peptide with numerous neuro-modulatory impacts beyond its role within the neuroendocrine system. TRH's broad central nervous system (CNS) effects, acting as a neurotransmitter and neuromodulator, emphasize a great potential to treat many neurological and psychological disorders. However, its pharmacological applications remain unrealized due to brain delivery shortcomings following its systemic administration. Previously, our laboratory's novel prodrug design, relying on two highly brain-expressed enzymes for prodrug metabolism to TRH, successfully delivered the metabolically highly unstable peptide into the brain. Consequently, an in vivo therapeutic safety assessment was conducted to further validate our prodrug approach through a comparative study that capitalizes on TRH's stimulatory release of thyroid hormones. Likewise, TRH's ability to trigger acetylcholine release is also well documented, and here, this neurochemical marker has been utilized to determine the extent to which TRH is delivered to the CNS via our prodrug approach. Moreover, our laboratory has recently identified pGlu-βGlu-Pro-NH2 ([βGlu2]TRH) as the first functional antagonist of the central cholinergic actions of TRH, and as such, we have explored the receptor-associated mechanism responsible for this antagonism by utilizing a human TRH receptor (hTRH-R) homology model. Ultimately, our extensive computational chemistry-based studies revealed a novel receptor allosteric site that exhibits a selective and high-affinity binding for [βGlu2 ]TRH, while also demonstrating our prodrug's inability to bind and activate this hTRH-R. Methods: Lead TRH prodrugs and various TRH analogues were designed in silico for docking experiments with the hTRH-R using SeeSAR and AutoDock Vina software. A TRH challenge in CD-1 mice, utilizing systemically administered TRH and an equimolar concentration of a TRH prodrug, measured downstream effector levels of thyroid hormones at several subsequent time points analyzed by LC-MS/MS. Microdialysis studies, in the frontal cortex of SD rats, compared each animal's baseline acetylcholine concentration to subsequent levels, following the perfusion of a TRH prodrug and TRH, as a positive control, at equimolar concentrations. This neurochemical survey quantifies acetylcholine turnover using LC-MS/MS, as a surrogate measure of the extent to which TRH is delivered into the brain via our prodrug approach. Results: Compared to TRH, prodrugs were unable to dock to the hTRH-R's active site, and when systemically administered, the TRH prodrug failed to elicit a thyroid response while simultaneously triggering a profound release of acetylcholine in the brain. Conclusions: The inability of TRH prodrugs to elicit a thyroid response was predicted by its in vitro metabolic stability, as well as computational chemistry studies, that demonstrate TRH prodrugs exhibit physiochemical properties that prohibit the direct activation of the hTRH-R. Furthermore, based on [βGlu2]TRH as a template, the design of novel hTRH-R inhibitors will be conducted in a follow-up study to further substantiate our prodrug approach and aid the elucidation of TRH activity and pathways.Item Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications(MDPI, 2021-02-08) Prokai, Laszlo; Rahlouni, Fatima; Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, KatalinThe widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17beta-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.Item PROTEOMICS-BASED DISCOVERY OF PROTEIN NETWORKS AND ASSOCIATED BIOLOGICAL PROCESSES IMPACTED BY ESTROGEN IN THE MALE RAT RETINA.(2022) Zaman, Khadiza; Nguyen, Vien; De La Cruz, Daniel; Prokai-Tatrai, Katalin; Prokai, LaszloPurpose: 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.Item Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma(MDPI, 2023-08-26) Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloElevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.Item Proteomics-Based Retinal Target Engagement Analysis and Retina-Targeted Delivery of 17beta-Estradiol by the DHED Prodrug for Ocular Neurotherapy in Males(MDPI, 2021-09-02) Prokai-Tatrai, Katalin; Zaman, Khadiza; Nguyen, Vien; De La Cruz, Daniel L.; Prokai, LaszloWe examined the impact of 17beta-estradiol (E2) eye drops on the modulation of the proteome profile in the male rat retina. With discovery-driven proteomics, we have identified proteins that were regulated by our treatment. These proteins were assembled to several bioinformatics-based networks implicating E2's beneficial effects on the male rat retina in a broad context of ocular neuroprotection including the maintenance of retinal homeostasis, facilitation of efficient disposal of damaged proteins, and mitochondrial respiratory chain biogenesis. We have also shown for the first time that the hormone's beneficial effects on the male retina can be constrained to this target site by treatment with the bioprecursor prodrug, DHED. A large concentration of E2 was produced after DHED eye drops not only in male rat retinae but also in those of rabbits. However, DHED treatment did not increase circulating E2 levels, thereby ensuring therapeutic safety in males. Targeted proteomics focusing on selected biomarkers of E2's target engagement further confirmed the prodrug's metabolism to E2 in the male retina and indicated that the retinal impact of DHED treatment was identical to that of the direct E2 treatment. Altogether, our study shows the potential of topical DHED therapy for an efficacious and safe protection of the male retina without the unwanted hormonal side-effects associated with current estrogen therapies.Item Retina-Targeted Delivery of 17beta-Estradiol by the Topically Applied DHED Prodrug(MDPI, 2020-05-16) Prokai-Tatrai, Katalin; Nguyen, Vien; De La Cruz, Daniel L.; Guerra, Rebecca; Zaman, Khadiza; Rahlouni, Fatima; Prokai, LaszloThe purpose of this study was to explore retina-targeted delivery of 17beta-estradiol (E2), a powerful neuroprotectant, by its bioprecursor prodrug 10beta,17beta-dihydroxyestra-1,4-dien-3-one (DHED) administered as eye drops in animal models. Compared to the parent hormone, DHED displayed increased transcorneal flux ex vivo both with and without the presence of 2-hydroxypropyl-beta-cyclodextrin used as a penetration-enhancing excipient in rat, rabbit, and pig. In vitro, the prodrug also showed facile bioactivation to E2 in the retina but not in the cornea. After topical administration to rats and rabbits, peak DHED-derived E2 concentrations reached 13 +/- 5 ng/g and 18 +/- 7 ng/g in the retina of female rats and rabbits, respectively. However, the prodrug remained inert in the rest of the body and, therefore, did not cause increase in circulating hormone concentration, as well as wet uterine and anterior pituitary weights as typical markers of E2's endocrine impact. Altogether, our studies presented here have demonstrated the premise of topical retina-selective estrogen therapy by the DHED prodrug approach for the first time and provide compelling support for further investigation into the full potential of DHED for an efficacious and safe ocular neurotherapy.Item ROAD MAPS TO GLAUCOMATOUS NEURODEGENERATION BY LABEL-FREE QUANTITATIVE PROTEOMICS(2021) Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloPurpose: There is a scarcity in reports on the molecular cross-talks occurring between the retina and optic nerve in glaucoma. Menopausal hormone deficiency also has been considered a risk factor of glaucomatous neurodegeneration hitherto without plausible mechanistic origins. To address these gaps of knowledge, we designed a global proteomics-based analysis of rat retina and optic nerve to gain a detailed understanding of IOP-associated alterations occurring at the protein level upon estrogen deprivation. Methods: Tissues were obtained through collaboration from rats in which one eye was subjected to routine IOP elevation by sclerosing the episcleral vein with hypertonic saline. Proteins from target tissues were extracted and analyzed separately for mass spectrometry-based proteomics using label-free quantification. Differentially expressed proteins were mapped to protein interaction networks and biological processes through Ingenuity Pathway Analysis (IPA?). Results: Our mass discovery-driven proteomic analysis covered nearly 1900 proteins in the rat retina and optic nerves. Regulated proteins triggered by increased IOP showed both distinct and similar functions between the retina and the optic nerve. The most interesting regulation we observed was the inhibition of signaling mechanisms involved in growth and proliferation, such as semaphorin signaling in neurons, and clathrin-mediated endocytosis responsible for maintaining cell morphology, regulating uptake of nutrients and synaptogenesis. Conclusion: Our study has provided the first global insight into alterations occurring at the molecular level and involving protein networks with associated biological processes upon glaucomatous neurodegeneration of the retina and optic nerve in response to elevated IOP.