Browsing by Author "Prokai-Tatrai, Katalin"
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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 A Novel Prodrug Approach for Central Nervous System-Selective Estrogen Therapy(MDPI, 2019-11-19) Prokai-Tatrai, Katalin; Prokai, LaszloBeneficial effects of estrogens in the central nervous system (CNS) results from the synergistic combination of their well-orchestrated genomic and non-genomic actions, making them potential broad-spectrum neurotherapeutic agents. However, owing to unwanted peripheral hormonal burdens by any currently known non-invasive drug administrations, the development of estrogens as safe pharmacotherapeutic modalities cannot be realized until they are confined specifically and selectively to the site of action. We have developed small-molecule bioprecursor prodrugs carrying the para-quinol scaffold on the steroidal A-ring that are preferentially metabolized in the CNS to the corresponding estrogens. Here, we give an overview of our discovery of these prodrugs. Selected examples are shown to illustrate that, independently of the route of administrations and duration of treatments, these agents produce high concentration of estrogens only in the CNS without peripheral hormonal liability. 10beta,17beta-Dihydroxyestra-1,4-dien-3-one (DHED) has been the best-studied representative of this novel type of prodrugs for brain and retina health. Specific applications in preclinical animal models of centrally-regulated and estrogen-responsive human diseases, including neurodegeneration, menopausal symptoms, cognitive decline and depression, are discussed to demonstrate the translational potential of our prodrug approach for CNS-selective and gender-independent estrogen therapy with inherent therapeutic safety.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 Advancing the Role of Thyrotropin-releasing Hormone as a Central Nervous System Agent(2022-05) De La Cruz, Daniel L.; Prokai-Tatrai, Katalin; Prokai, Laszlo; Lacko, Andras G.; Forster, Michael J.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 Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes(-)4(MDPI, 2018-12-31) Mangoni, Arduino A.; Guillou, Catherine; Vanden Eynde, Jean Jacques; Hulme, Christopher; Jampilek, Josef; Li, Wei; Prokai-Tatrai, Katalin; Rautio, Jarkko; Collina, Simona; Tuccinardi, Tiziano; Sousa, Maria Emilia; Sabatier, Jean-Marc; Galdiero, Stefania; Karaman, Rafik; Kokotos, George; Torri, Giangiacomo; Luque, F. Javier; Vasconcelos, M. Helena; Hadjipavlou-Litina, Dimitra; Siciliano, Carlo; Gutschow, Michael; Ragno, Rino; Gomes, Paula A. C.; Agrofoglio, Luigi A.; Munoz-Torrero, DiegoBreakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials, which is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules. [...].Item Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-3(MDPI, 2018-06-30) Mangoni, Arduino A.; Tuccinardi, Tiziano; Collina, Simona; Vanden Eynde, Jean Jacques; Munoz-Torrero, Diego; Karaman, Rafik; Siciliano, Carlo; de Sousa, Maria Emilia; Prokai-Tatrai, Katalin; Rautio, Jarkko; Guillou, Catherine; Gutschow, Michael; Galdiero, Stefania; Liu, Hong; Agrofoglio, Luigi A.; Sabatier, Jean-Marc; Hulme, Christopher; Kokotos, George; You, Qidong; Gomes, Paula A. C.Item Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-5(MDPI, 2019-06-30) Mangoni, Arduino A.; Eynde, Jean Jacques Vanden; Jampilek, Josef; Hadjipavlou-Litina, Dimitra; Liu, Hong; Reynisson, Johannes; Sousa, Maria Emilia; Gomes, Paula A. C.; Prokai-Tatrai, Katalin; Tuccinardi, Tiziano; Sabatier, Jean-Marc; Luque, F. Javier; Rautio, Jarkko; Karaman, Rafik; Vasconcelos, M. Helena; Gemma, Sandra; Galdiero, Stefania; Hulme, Christopher; Collina, Simona; Gutschow, Michael; Kokotos, George; Siciliano, Carlo; Capasso, Raffaele; Agrofoglio, Luigi A.; Ragno, Rino; Munoz-Torrero, DiegoBreakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials which is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules [...].Item Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-6(MDPI, 2019-12-28) Vanden Eynde, Jean Jacques; Mangoni, Arduino A.; Rautio, Jarkko; Leprince, Jerome; Azuma, Yasu-Taka; Garcia-Sosa, Alfonso T.; Hulme, Christopher; Jampilek, Josef; Karaman, Rafik; Li, Wei; Gomes, Paula A. C.; Hadjipavlou-Litina, Dimitra; Capasso, Raffaele; Geronikaki, Athina; Cerchia, Laura; Sabatier, Jean-Marc; Ragno, Rino; Tuccinardi, Tiziano; Trabocchi, Andrea; Winum, Jean-Yves; Luque, F. Javier; Prokai-Tatrai, Katalin; Spetea, Mariana; Gutschow, Michael; Kosalec, Ivan; Guillou, Catherine; Vasconcelos, M. Helena; Kokotos, George; Rastelli, Giulio; de Sousa, Maria Emilia; Manera, Clementina; Gemma, Sandra; Mangani, Stefano; Siciliano, Carlo; Galdiero, Stefania; Liu, Hong; Scott, Peter J. H.; de Los Rios, Cristobal; Agrofoglio, Luigi A.; Collina, Simona; Guedes, Rita C.; Munoz-Torrero, DiegoBreakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials that is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules [...].Item Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-7(MDPI, 2020-06-28) Gutschow, Michael; Eynde, Jean Jacques Vanden; Jampilek, Josef; Kang, CongBao; Mangoni, Arduino A.; Fossa, Paola; Karaman, Rafik; Trabocchi, Andrea; Scott, Peter J. H.; Reynisson, Johannes; Rapposelli, Simona; Galdiero, Stefania; Winum, Jean-Yves; Brullo, Chiara; Prokai-Tatrai, Katalin; Sharma, Arun K.; Schapira, Matthieu; Azuma, Yasu-Taka; Cerchia, Laura; Spetea, Mariana; Torri, Giangiacomo; Collina, Simona; Geronikaki, Athina; Garcia-Sosa, Alfonso T.; Vasconcelos, M. Helena; Sousa, Maria Emilia; Kosalec, Ivan; Tuccinardi, Tiziano; Duarte, Iola F.; Salvador, Jorge A. R.; Bertinaria, Massimo; Pellecchia, Maurizio; Amato, Jussara; Rastelli, Giulio; Gomes, Paula A. C.; Guedes, Rita C.; Sabatier, Jean-Marc; Estevez-Braun, Ana; Pagano, Bruno; Mangani, Stefano; Ragno, Rino; Kokotos, George; Brindisi, Margherita; Gonzalez, Florenci V.; Borges, Fernanda; Miloso, Mariarosaria; Rautio, Jarkko; Munoz-Torrero, DiegoBreakthroughs in Medicinal Chemistry [...].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 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 Improving Brain Delivery of TRH: A Novel Prodrug Approach(2018-08-01) De La Cruz, Daniel L.; Prokai-Tatrai, Katalin; Prokai, Laszlo; Lacko, Andras G.The goal of my research project was to validate a novel prodrug design concept for the brain-enhanced delivery of an important neuropeptide, thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2). TRH has a variety of clinically relevant central effects that cannot be utilized with the direct administration of TRH, due to intrinsic characteristics that give rise to metabolic instability and insufficient transport through the blood-brain barrier (BBB). Consequently, large doses are required to generate central effects, which concomitantly induces unwanted hormonal liabilities in the periphery. To overcome these caveats, the prodrug design described herein proposes a novel brain-targeting approach that synergistically utilizes two enzymes, both preferentially expressed in the brain, for the enhanced brain-delivery of TRH. The conjugation of a lipoamino acid (LAA) transport moiety to the N-terminus of a TRH progenitor sequence (Gln-His-Pro-NH2) via a prolyl oligopeptidase (POP)-sensitive linker allows the POP enzyme to release the TRH progenitor sequence at the site-of-action upon crossing the BBB, where it is further transformed by glutaminyl cyclase (QC). QC catalyzes Gln to form pGlu at the N-terminus of the progenitor sequence, thereby releasing TRH. I tested the hypothesis that a representative molecule, developed according to this prodrug design approach, would exhibit adequate drug-likeness for BBB penetration and efficacious release of TRH within the brain. Immobilized artificial membrane chromatography was used to predict the BBB penetration of this experimental prodrug, labeled “PRODRUG 1,” in addition to its calculated logP. Next, PRODRUG (1) was compared to TRH (the “parent” peptide) in an in vitro metabolic stability assessment, followed by an in vivo neuro-pharmacodynamical evaluation in rodents. The Porsolt swim test, PST, an established animal behavioral model that detects depressive-like behavior was used to confirm brain-delivery of prodrug-derived TRH after systemic administration of the prototype prodrug. Capitalizing on TRH’s antidepressant-like effect, the PST results were also used to validate the tail suspension test (TST), a new technique that I implemented in our laboratory for the evaluation of neuroactive compounds with potential antidepressant-like activity. My findings support the extension of the TRH progenitor sequence from the N-terminus, through the conjugation of two LAA residues (each with a 10-carbon sidechain) via a single proline POP-sensitive linker, as a successful means to increase penetration across the BBB and sufficiently bind with cleaving and activating enzymes, POP and QC, respectively, for efficacious TRH release in the brain. Lastly, molecular modeling was used to create a library of similarly designed prodrugs to computationally assess their bindings with POP, the cleaving enzyme, to further explore the customizable prodrug design concept described here. Ultimately, this adaptable prodrug delivery model demonstrates the effectiveness of increased lipophilicity and site-of-action targeting to facilitate brain-enhanced delivery of TRH.Item Investigating the Effect of 17β-Estradiol on 24(S)-hydroxycholesterol Concentration in Brain Tissue(2021-05) Oaxaca, K. Celeste; Prokai, Laszlo; Prokai-Tatrai, Katalin; Lacko, Andras G.; Woerner, August E.Estrogen, more specifically 17β-estradiol (E2), has been found to have neuroprotective effects in the brain. With Alzheimer's patients being comprised mainly of women, it is worthwhile to investigate the role that reproductive senescence plays in the development and progression of the disease due to decreased levels of estrogen. The purpose of this research was to develop a method for analyzing how estrogen affects the concentration of 24(S)-hydroxycholesterol (24S-OHC) in brain tissue. Ovariectomized (OVX) CD1 mice we subjected to daily injections of E2 for 5 days. Cholesterol and 24SOHC from hippocampal tissue were analyzed by gas chromatography-mass spectrometry (GC-MS) assay developed for the purpose, and their concentrations were compared between the OVX E2 treated and OVX control groups. No significant difference was found between the two groups; nevertheless, the assay developed during this study will be beneficial for further investigation of E2 as a potential modulator of cholesterol metabolism in future studies.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.
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