Browsing by Author "Nguyen, Vien"
Now showing 1 - 20 of 27
- Results Per Page
- Sort Options
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 A VALIDATED METHOD FOR BIOANALYSIS OF ACETYLCHOLINE FROM CEREBROSPINAL FLUIDS USING LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY(2021) Nguyen, Vien; De La Cruz, Daniel; Prokai-Tatrai, Katalin; Prokai, LaszloDecreased acetylcholine [ACh, (CH3)3N+CH2CH2OCOCH3] levels have been associated with several diseases of the central nervous system (CNS) such as Alzheimer's disease; therefore, it has been considered a potential biomarker. However, quantification of ACh in biological samples even in cerebrospinal fluids obtained through proximity sampling is difficult due to its often extremely low concentration owing to rapid enzyme-catalyzed hydrolysis to choline. Most limitations of previously used liquid chromatography-based (LC) assays relying on electrochemical detection have been addressed by coupling LC with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). However, numerous applications have remained challenging because of inadequate detection sensitivity achievable with most commercial instruments and columns. We improved the method using atmospheric pressure spray ionization (APSI) as an alternative to ESI and ultrahigh-performance liquid chromatography (UPLC) resulting in an about 100-fold increase in detection sensitivity over the previous LC–ESI-MS/MS assay. The presentation will feature application of the method to the sensitive determination of ACh in rodent brain microdialysates, which indicates fit for purpose to use in translational research focusing on this important neurotransmitter. The developed assay also has been rigorously validated to show that it would meet regulatory requirements for application to assess ACh as a potential clinical biomarker of CNS diseases.Item Analysis of Acetylcholine in Cerebrospinal Fluids by Liquid Chromatography–Tandem Mass Spectrometry(2019-03-05) Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, Laszlo; Ochoa, SamanthaAnalysis of Acetylcholine in Cerebrospinal Fluids by Liquid Chromatography–Tandem Mass Spectrometry Samantha M. Brewer, Vien Nguyen, Katalin Prokai-Tatrai and Laszlo Prokai Department of Pharmacology and Neuroscience, Graduate School of Biomedical Sciences Purpose: Acetylcholine (ACh) is a neurotransmitter whose decreased levels have been associated with several diseases impacting the central nervous system. Quantification of extracellular ACh in cerebrospinal fluid (CSF) is difficult due to its low concentration (owing to its rapid hydrolysis to choline) and matric effects upon employing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification. The purpose of this study was to develop and validate an LC-MS/MS method for the high-throughput determination of ACh in rat brain microdialysates and possibly in clinical samples in the future. Methods: All analyses were performed on a TSQ Quantum Ultra mass spectrometer connected to a Surveyor MS high-performance liquid chromatography system (Thermo Scientific, San Jose, CA). Atmospheric spray ionization was used, and analytes were detected after fragmenting their intact cations through collision-induced dissociation. Deuterium-labeled ACh (ACh-d4) was used as an internal standard for quantification. Several solvent systems were compared to identify the best conditions for the preparation of ACh and ACh-d4 stock solutions to calibrate the assay. The optimized method was used to determine the extracellular ACh levels in rat brain microdialysates using the calibration curve obtained in aCSF. Results: In order to set-up a reliable assay, the MS signals for ACh and deuterium-labeled ACh (ACh-d4) used as an internal standard had to be optimized, along with optimizing the solvent systems for the preparation of their stock solutions. The optimal solvent system afforded linear calibration and resisted signal suppression by artificial cerebrospinal fluid (aCSF). Artificial cerebrospinal fluid is used as perfusion fluid upon microdialysis as it matches the composition of actual CSF. We have established that the use of purely aqueous stock solutions of ACh and ACh-d4 afforded the best calibration in aCSF. With these measures, reliable analyses of ACh in aCSF were achieved. The obtained linear calibration could be used for quantitation of low (physiological) ACh levels in samples obtained upon in vivo intracranial microdialysis from rat cortex. Conclusions: The method presented here allows for reliable quantification of ACh in cerebrospinal fluids, and may be used to evaluate the efficiency and mode of action of potential cholinergic agents. Acknowledgment: This research has been supported in part by The Welch Foundation (endowment BK-0031). IACUC Number: 2018-0006Item [beta-Glu(2)]TRH Is a Functional Antagonist of Thyrotropin-Releasing Hormone (TRH) in the Rodent Brain(MDPI, 2021-06-09) Prokai-Tatrai, Katalin; Nguyen, Vien; Prokai, LaszloSelective antagonists of thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), in order to enable a better understanding of this peptide's central functions, have not been identified. Using pGlu-Glu-Pro-NH2 ([Glu(2)]TRH) as a lead peptide and with modification at its central residue, our studies focused on some of its analogues synthesized as potential functional antagonists of TRH in the rodent brain. Among the peptides studied, the novel isomeric analogue [beta-Glu(2)]TRH was found to suppress the analeptic and antidepressant-like pharmacological activities of TRH without eliciting intrinsic effects in these paradigms. [beta-Glu(2)]TRH also completely reversed TRH's stimulation of acetylcholine turnover in the rat hippocampus without a cholinergic activity of its own, which was demonstrated through in vivo microdialysis experiments. Altogether, [beta-Glu(2)]TRH emerged as the first selective functional antagonist of TRH's prominent cholinergic actions, by which this endogenous peptide elicits a vast array of central effects.Item Brain Delivery of Thyrotropin-Releasing Hormone via a Novel Prodrug Approach(MDPI, 2019-07-18) Prokai-Tatrai, Katalin; De La Cruz, Daniel L.; Nguyen, Vien; Ross, Benjamin P.; Toth, Istvan; Prokai, LaszloUsing thyrotropin-releasing hormone (TRH) as a model, we explored whether synergistic combination of lipoamino acid(s) and a linker cleaved by prolyl oligopeptidase (POP) can be used as a promoiety for prodrug design for the preferential brain delivery of the peptide. A representative prodrug based on this design principle was synthesized, and its membrane affinity and in vitro metabolic stability, with or without the presence of a POP inhibitor, were studied. The in vivo formation of TRH from the prodrug construct was probed by utilizing the antidepressant effect of the peptide, as well as its ability to increase acetylcholine (ACh) synthesis and release. We found that the prototype prodrug showed excellent membrane affinity and greatly increased metabolic stability in mouse blood and brain homogenate compared to the parent peptide, yet a POP inhibitor completely prevented prodrug metabolism in brain homogenate. In vivo, administration of the prodrug triggered antidepressant-like effect, and microdialysis sampling showed greatly increased ACh release that was also antagonized upon a POP inhibitor treatment. Altogether, the obtained promising exploratory data warrant further investigations on the utility of the prodrug approach introduced here for brain-enhanced delivery of small peptides with neurotherapeutic potential.Item Brain-Selective Estrogen Therapy Prevents Androgen Deprivation-Associated Hot Flushes in a Rat Model(MDPI, 2020-06-10) Merchenthaler, Istvan; Lane, Malcolm; Stennett, Christina; Zhan, Min; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloHot flushes are best-known for affecting menopausal women, but men who undergo life-saving castration due to androgen-sensitive prostate cancer also suffer from these vasomotor symptoms. Estrogen deficiency in these patients is a direct consequence of androgen deprivation, because estrogens (notably 17beta-estradiol, E2) are produced from testosterone. Although estrogens alleviate hot flushes in these patients, they also cause adverse systemic side effects. Because only estrogens can provide mitigation of hot flushes on the basis of current clinical practices, there is an unmet need for an effective and safe pharmacotherapeutic intervention that would also greatly enhance patient adherence. To this end, we evaluated treatment of orchidectomized (ORDX) rats with 10beta, 17beta-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective bioprecursor prodrug of E2. A pilot pharmacokinetic study using oral administration of DHED to these animals revealed the formation of E2 in the brain without the appearance of the hormone in the circulation. Therefore, DHED treatment alleviated androgen deprivation-associated hot flushes without peripheral impact in the ORDX rat model. Concomitantly, we showed that DHED-derived E2 induced progesterone receptor gene expression in the hypothalamus without stimulating galanin expression in the anterior pituitary, further indicating the lack of systemic estrogen exposure upon oral treatment with DHED.Item Brain-Targeting Prodrug Design for Thyrotropin Releasing Hormone(2019-03-05) Nguyen, Vien; Prokai, Laszlo; Prokai-Tatrai, Katalin; Del La Cruz, DanielPurpose: Thyrotropin releasing hormone (TRH) has many neuromodulatory effects throughout the brain, however, treatment using this peptide induces unwanted peripheral side-effects. Based on our novel prodrug design that synergistically employs lipoamino acid residues (LAAs) and a brain-enzyme sensitive linker for prolyl oligopeptidase (POP), we have developed a set of lead compounds in silico. Computationally assessing lipophilicity and POP-binding affinity of our virtual prodrugs led to the selection of a representative, termed Prodrug (1), for membrane affinity studies to predict brain access from circulation. Method: Prodrugs with different LAAs and POP-sensitive linkers were designed in silico for docking with POP’s binding site using SCIGRESS and AutoDock Vina software. The prodrugs’ calculated logP (clogP) and POP-binding affinity were determined with built-in SCIGRESS applications. AutoDock results were represented as Gibbs free energy of binding (ΔG). The molecules with the most negative ΔG and adequate clogP were selected for synthesis. Prodrug (1) was compared to TRH in membrane affinity studies via immobilized artificial membrane chromatography (IAMC), an established method to predict membrane affinity (i.e., BBB permeability) with a chromatographic column comprised of immobilized synthetic lipids that mimic biological membranes. A high IAMC retention time correlates to a greater membrane affinity, with IAM Chromatographic Hydrophobicity Index (CHIIAM) values being the quantitative measure of retention. A range of reference compounds with known CHIIAM values were selected; plotting these values against their experimentally determined gradient retention times through the IAM column provided the linear relationship equation used to convert Prodrug (1)’s gradient retention time into its CHIIAM value. Results: A virtual library of prodrugs having various LAAs and POP-sensitive linkers were designed and docked to POP’s binding site. The binding of Prodrug (1) was comparable to the calculated ΔG of POP’s published, co-crystalized ligand. This prodrug also showed favorable clogP for transport into the brain and a significantly increased CHIIAM compared to TRH. Conclusion: Based on the prediction of POP-binding, using in silico docking and the favorable membrane affinity of Prodrug (1), we expect this prodrug to efficiently deliver TRH into the brain and serve as a template for fine-tuning future prodrug constructs for the efficacious brain-delivery of TRH.Item Computer-Assisted Design and In Silico Screening of Brain-Targeting Peptide Prodrugs(2020) Prokai, Laszlo; Prokai-Tatrai, Katalin; Nguyen, Vien; De La Cruz, DanielPurpose: Prodrug approaches promise overcoming obstacles for the brain delivery of therapeutically useful small neuropeptides. However, their rational design and large-scale computational screening have been elusive. Recently, we have introduced a method that not only enhances a prodrug's access to the brain through lipoamino acid residues (LAAs), but also ensures site-specific bioactivation by prolyl oligopepdidase (POP) selectively expressed in neuronal milieu. Using thyrotropin-releasing hormone (TRH) as a model, we show a computer-aided strategy to assist the design and optimization of brain-targeting prodrugs for small neuropeptides. Methods: Using SCIGRESS molecular modeling and AutoDock Vina docking, lipophilicity (indicated by calculated logarithm of octanol/water partition coefficient, clogP) and binding affinity to POP (expressed as free energy change, ΔG) were assessed in silico for a virtual library of prodrugs. A computational model of human TRH receptor (hTRHr-1) was also adapted to provide proof of concept for the prodrug principle. Results: Prodrugs of a virtual library docked computationally to POP's active site displayed ΔG values comparable to that of a co-crystallized POP ligand, but with subtle differences based on the configuration of the LAAs and the POP-sensitive linker. Expectedly, they showed no affinity for binding to hTRHr-1's active site. Co-optimization based on clogP has allowed for the selection of top TRH prodrug candidates for further in vitro and in vivo evaluations. Conclusion: We have created a comprehensive in silico workflow to aid the rational design and large-scale virtual screening of brain-targeting neuropeptide prodrugs.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 Effect of estradiol replacement on hippocampal concentrations of estrogens in aged rhesus macaques maintained on an obesogenic diet(Elsevier B.V., 2023-09-25) Prokai, Laszlo; Nguyen, Vien; Urbanski, Henryk F.Replacement involving estrogens has proven efficacy at treating a wide range of disorders that develop with menopause or after surgical removal of the ovaries. Here, we tested whether an estradiol (E2) replacement paradigm that recapitulates physiological E2 levels in the circulation also recapitulates physiological E2 levels within the hippocampus. E2 was delivered continuously to old ovariectomized (OVX) rhesus macaques, maintained on a high-fat, high-sugar Western-style diet (WSD) for approximately 30 months, via subcutaneous implants; this resulted in physiological concentrations of both estrone (E1) and E2 in the circulation (determined by LC-MS/MS). Surprisingly, however, hippocampal concentrations of E2 were markedly (P < 0.01) higher than in ovary-intact animals maintained on a regular chow diet. The data suggest that E2 replacement paradigms that appear to recapitulate physiological E2 concentrations in the circulation may produce hyper-physiological E2 levels within some brain areas, especially when individuals are maintained on a WSD.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 Neuropharmacological Responses are Observed in Mice After Systemic Treatment with a Prodrug of Thyrotropin-Releasing Hormone(2016-03-23) Bailey, Brooke; Nguyen, Vien; Prokai, Laszlo; Prokai-Tatrai, Katalin; Shurlknight, KellyShort description: Thyrotrophin-releasing hormone (TRH) is a promising agent to treat various brain maladies. However, delivery of this peptide into the brain has been an obstacle. In this exploratory study, a TRH prodrug designed for brain-enhanced delivery was evaluated through the reversal of drug-induced narcosis (analeptic effect) and antidepressant-like effect. Purpose: To show the utility of ketamine/xylazine in a mouse model to evaluate analeptic effect evoked by TRH upon its brain-delivery via a novel prodrug approach, as well as to confirm the antidepressant-like effect of this neuroactive peptide when delivered into the brain. Methods: For the assessment of analeptic effect, CD1 mice were divided into groups of n = 8. Test compounds were dissolved in saline. Mice were treated with a single dose (10 µmol/kg) of prodrug, saline vehicle, TRH, respectively, through the tail vein by i.v. injections. Ten minutes after injection of drug, mice were injected i.p. with either a mixture of ketamine (100 mg/kg) and xylazine (10mg/kg), or sodium pentobarbital (60 mg/kg). Sleeping time was recorded starting from the loss of righting reflex until this reflex was regained. To evaluate antidepressant-like activity using the Porsolt’s swim test, test compounds were administered i.v. through the tail vein at the dose of 3 µmol/kg, in a separate study. For 6 min, the immobility time (the duration of motionless floating after the cessation of struggling and making only movements necessary to keep the head above the water) was recorded. Results: TRH also reduced sleeping times after ketamine/xylazine sedation. Administration of the TRH prodrug also manifested analeptic effect characteristic to the parent peptide. Like after TRH injection, immobility time in Porsolt’s swim test indicative of the peptide’s antidepressant-like activity also shortened after the administration of its prodrug when compared to saline control. Thus, the analeptic and antidepressant-like effects observed after systemic administration of the TRH prodrug has reflected its ability to penetrate the blood-brain barrier followed by the release of the parent peptide at the site of action. Delivery of TRH to the brain by specific prodrug approach would allow for reduction of TRH’s endocrine side effects and for a prolonged duration of action. Conclusions: Treatment by a TRH prodrug reduced the ketamine/xylazine- and sodium pentobarbital-induced sleeping time in mice, as well as shortened the immobility time in Porsolt’s swim test. These observations have indicated a successful delivery of the neuroactive peptide into the brain via its prodrug introduced here.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 Non-Feminizing Estrogens Do Not Exhibit Antidepressant-Like Activity(2016-03-23) Prokai, Laszlo; Prokai-Tatrai, Katalin; Nguyen, VienShort description: In this exploratory lead compound evaluation, we aimed at addressing the utility of two non-feminizing estrogens, specifically 2-adamantyl-17β-estradiol (Ada-E2) and 2-adamantylestrone (Ada-E1), in a well-established animal model of depression-like behavior precipitated by estrogen deprivation. Purpose: To evaluate non-feminizing estrogens in fulfilling their overall premise for the treatment of climacteric symptoms. Methods: Mice were divided into six animals per treatment group. Test agents were dissolved either in corn oil vehicle or in 30% v/v aqueous 2-hydroxypropyl-β-cyclodextrin. The well-known antidepressant amitriptyline, as a reference standard, was used at 15 mg/kg dose, while the estrogen receptor (ER) antagonist fulvestrant was used at 4 mg/kg dose. The control groups received vehicle only. Test compounds in corn oil vehicle were administered subcutaneously (s.c.), while those in HPβCD were given intravenously (i.v.). Each group of animals was treated daily for five consecutive days injecting the test agents 100 µg/kg or 500 µg/kg doses on each day. Antidepressant-like activity were evaluated 30 min after the last injection using the Porsolt swim test (PST). The immobility time (in seconds, defined as the duration of floating motionless after the cessation of struggling and making only movements necessary to keep the head above the water) was recorded for 6 min simultaneously by a trained observer. Drug-likeness was evaluated via the online Osiris Property Explorer. Antioxidant potencies were determined experimentally by the ferric thiocyanate and thiobarbituric acid reactive substances methods. Results: Adding the bulky Ada to the already lipophilic E2 and E1 brought about further increase in the lipophilicity (logP) by [greater than] 2 log units. This increase was probably the most profound contributor to their unfavorable drug-likeness score. In agreement with our earlier quantitative structure–activity relationship study, our experimental assessment also supported that an increase in logP enhances antioxidant effect of estrogen-derived synthetic steroids and their analogs. However, while E2 and E1 did show significant reduction of immobility time in the mice PST, Ada-E2 and Ada-E1 failed to manifest activity in this paradigm (Immobility time is associated with depression-like behavior). Therefore, ERs play a pivotal role in triggering depression-like behavior in estrogen-deprived animals and non-feminizing estrogen offers no remedy for this symptom. Conclusions: Our lead evaluation has confirmed that both genomic and non-genomic mechanisms are required for broad-spectrum estrogen neuroprotection and treatment of menopausal symptoms. Therefore, non-feminizing estrogens such as Ada-E2 and Ada-E1 are not appropriate for the management of symptoms that manifest through ERs such as depression.Item Pilot Study Using the Tail Suspension Test(2018-03-14) Del La Cruz, Daniel; Nguyen, Vien; Prokai-Tatrai, Katalin; Berihun, ElizabethPurpose: The Porsolt Swim Test (PST) is an established protocol to study the antidepressant-like effect of potential CNS agents. The purpose of this study is to introduce a complimentary behavioral model to assess this central effect; specifically, in this study, we implemented the Tail Suspension Test (TST) to our lab. The validation of the TST model was performed by exploiting the well-known antidepressant-like effect of thyrotropin releasing hormone (TRH) in a comparative fashion with that of the PST. Method: We constructed an apparatus to perform the TST where mice are suspended by their tails. The motionless hanging of an animal is associated with a depressive state, and the time an animal spends in this state is called immobility time. In the PST, on the other hand, an animal is placed in an inescapable water-filled cylinder, and immobility time is defined as when an animal only makes minimal movements to keep its head above the water. To validate the newly introduced TST, CD1 mice received either vehicle or various concentrations of TRH via dorsal s.c. injection 30 min before testing in both paradigms. Two independent observers measured immobility time for the duration of the experiment, after which animals were sacrificed and tissues harvested for future TRH quantification. Mice were only exposed to these experimental conditions on the day of the experiment and each mouse was only subjected to a single dose and subsequent test -no preconditioning was required. Results: Comparisons of the TRH-treated groups and the control group showed a statistically significant difference in immobility time, indicating the antidepressant-like effect of this small neuropeptide. A dose-dependent reduction of immobility time was observed in both the PST and the TST; moreover, the calculated ED50 values were not significantly different from each other. Specifically, ED50 was approximated at 2 µmol/kg body weight in the PST, and about 1.5 µmol/kg body weight in the TST. Conclusion: We have successfully implemented the TST, a complimentary paradigm to the PST, to study potential antidepressant-like agents. Moreover, we have also shown that TRH, a neuropeptide with well-known antidepressant effect exhibits similar potency in both experimental paradigms.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.