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Aladev SD, Sokolov DK, Strokotova AV, Kazanskaya GM, Volkov AM, Aidagulova SV, Grigorieva EV. Multiple Administration of Dexamethasone Possesses a Deferred Long-Term Effect to Glycosylated Components of Mouse Brain. Neurol Int 2024; 16:790-803. [PMID: 39051219 PMCID: PMC11270268 DOI: 10.3390/neurolint16040058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Revised: 07/13/2024] [Accepted: 07/18/2024] [Indexed: 07/27/2024] Open
Abstract
Glucocorticoids are used during glioblastoma treatment to prevent the cerebral edema effect surrounding normal brain tissue. The aim of our study was to investigate the long-term effects of multiple administrations of glucocorticoids onto the glycosylated components (proteoglycans and glycosaminoglycans) of normal brain extracellular matrix and the glucocorticoid receptor (GR, Nr3c1) in an experimental model in vivo. Two-month-old male C57Bl/6 mice (n = 90) were injected intraperitoneally with various doses of dexamethasone (DXM) (1; 2.5 mg/kg) for 10 days. The mRNA levels of the GR, proteoglycans core proteins, and heparan sulfate metabolism-involved genes were determined at the 15th, 30th, 60th, and 90th days by a real-time RT-PCR. The glycosaminoglycans content was studied using dot blot and staining with Alcian blue. A DXM treatment increased total GAG content (2-fold), whereas the content of highly sulfated glycosaminoglycans decreased (1.5-2-fold). The mRNA level of the heparan sulfate metabolism-involved gene Hs3St2 increased 5-fold, the mRNA level of Hs6St2 increased6-7-fold, and the mRNA level of proteoglycan aggrecan increased 2-fold. A correlation analysis revealed an association between the mRNA level of the GR and the mRNA level of 8 of the 14 proteoglycans-coding and 4 of the 13 heparan sulfate metabolism-involved genes supporting GR involvement in the DXM regulation of the expression of these genes. In summary, multiple DXM administrations led to an increase in the total GAG content and reorganized the brain extracellular matrix in terms of its glycosylation pattern.
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Affiliation(s)
- Stanislav D. Aladev
- Institute of Molecular Biology and Biophysics FRC FTM, Novosibirsk 630117, Russia; (D.K.S.); (A.V.S.); (G.M.K.); (S.V.A.); (E.V.G.)
| | - Dmitry K. Sokolov
- Institute of Molecular Biology and Biophysics FRC FTM, Novosibirsk 630117, Russia; (D.K.S.); (A.V.S.); (G.M.K.); (S.V.A.); (E.V.G.)
| | - Anastasia V. Strokotova
- Institute of Molecular Biology and Biophysics FRC FTM, Novosibirsk 630117, Russia; (D.K.S.); (A.V.S.); (G.M.K.); (S.V.A.); (E.V.G.)
| | - Galina M. Kazanskaya
- Institute of Molecular Biology and Biophysics FRC FTM, Novosibirsk 630117, Russia; (D.K.S.); (A.V.S.); (G.M.K.); (S.V.A.); (E.V.G.)
- E.N. Meshalkin National Medical Research Center, Novosibirsk 630055, Russia;
| | - Alexander M. Volkov
- E.N. Meshalkin National Medical Research Center, Novosibirsk 630055, Russia;
| | - Svetlana V. Aidagulova
- Institute of Molecular Biology and Biophysics FRC FTM, Novosibirsk 630117, Russia; (D.K.S.); (A.V.S.); (G.M.K.); (S.V.A.); (E.V.G.)
- Laboratory of Cellular Biology, Novosibirsk State Medical University, Novosibirsk 630091, Russia
| | - Elvira V. Grigorieva
- Institute of Molecular Biology and Biophysics FRC FTM, Novosibirsk 630117, Russia; (D.K.S.); (A.V.S.); (G.M.K.); (S.V.A.); (E.V.G.)
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Naeini SE, Bhandari B, Gouron J, Rogers HM, Chagas PS, Naeini GE, Chagas HIS, Khodadadi H, Salles ÉL, Seyyedi M, Yu JC, Grochowska BK, Wang LP, Baban B. Reprofiling synthetic glucocorticoid-induced leucine zipper fusion peptide as a novel and effective hair growth promoter. Arch Dermatol Res 2024; 316:190. [PMID: 38775976 DOI: 10.1007/s00403-024-02988-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2024] [Revised: 04/25/2024] [Accepted: 04/26/2024] [Indexed: 05/30/2024]
Abstract
Hair is a biofilament with unique multi-dimensional values. In human, in addition to physiologic impacts, hair loss and hair related disorders can affect characteristic features, emotions, and social behaviors. Despite significant advancement, there is a dire need to explore alternative novel therapies with higher efficacy, less side effects and lower cost to promote hair growth to treat hair deficiency. Glucocorticoid-induced leucine zipper (GILZ) is a protein rapidly induced by glucocorticoids. Studies from our group and many others have suggested that a synthetic form of GILZ, TAT-GILZ, a fusion peptide of trans-activator of transcription and GILZ, can function as a potent regulator of inflammatory responses, re-establishing and maintaining the homeostasis. In this study, we investigate whether TAT-GILZ could promote and contribute to hair growth. For our pre-clinical model, we used 9-12 week-old male BALB/c and nude (athymic, nu/J) mice. We applied TAT-GILZ and/or TAT (vehicle) intradermally to depilated/hairless mice. Direct observation, histological examination, and Immunofluorescence imaging were used to assess the effects and compare different treatments. In addition, we tested two current treatment for hair loss/growth, finasteride and minoxidil, for optimal evaluation of TAT-GILZ in a comparative fashion. Our results showed, for the first time, that synthetic TAT-GILZ peptide accelerated hair growth on depilated dorsal skin of BALB/c and induced hair on the skin of athymic mice where hair growth was not expected. In addition, TAT-GILZ was able to enhance hair follicle stem cells and re-established the homeostasis by increasing counter inflammatory signals including higher regulatory T cells and glucocorticoid receptors. In conclusion, our novel findings suggest that reprofiling synthetic TAT-GILZ peptide could promote hair growth by increasing hair follicle stem cells and re-establishing homeostasis.
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Affiliation(s)
- Sahar Emami Naeini
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Bidhan Bhandari
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Jules Gouron
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Hannah M Rogers
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Pablo Shimaoka Chagas
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Golnaz Emami Naeini
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Henrique Izumi Shimaoka Chagas
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Hesam Khodadadi
- Department of Neurology, Medical College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Évila Lopes Salles
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Mohammad Seyyedi
- Piedmont Ear, Nose, Throat and Related Allergy, Atlanta, GA, USA
| | - Jack C Yu
- Department of Surgery, Medical College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | | | - Lei P Wang
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Babak Baban
- DCG Center for Excellence in Research, Scholarship and Innovation (CERSI), Augusta University, Augusta, GA, USA.
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University, Augusta, GA, 30912, USA.
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Gallo MT, Dolci B, Fumagalli F, Brivio P, Calabrese F. Prenatal Fluoxetine Exposure Influences Glucocorticoid Receptor-Mediated Activity in the Prefrontal Cortex of Adolescent Rats Exposed to Acute Stress. ACS Chem Neurosci 2024; 15:1560-1569. [PMID: 38507566 DOI: 10.1021/acschemneuro.3c00856] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/22/2024] Open
Abstract
Any deviation from the programmed processes of brain development may modify its formation and functions, thereby precipitating pathological conditions, which often become manifest in adulthood. Exposure to a challenge during crucial periods of vulnerability, such as adolescence, may reveal molecular changes preceding behavioral outcomes. Based on a previous study showing that prenatal fluoxetine (FLX) leads to the development of an anhedonic-like behavior in adult rats, we aimed to assess whether the same treatment regimen (i.e., fluoxetine during gestation; 15 mg/kg/day) influences the ability to respond to acute restraint stress (ARS) during adolescence. We subjected the rats to a battery of behavioral tests evaluating the development of various phenotypes (cognitive deficit, anhedonia, and anxiety). Furthermore, we carried out molecular analyses in the plasma and prefrontal cortex, a brain region involved in stress response, and whose functions are commonly altered in neuropsychiatric conditions. Our findings confirm that prenatal manipulation did not affect behavior in adolescent rats but impaired the capability to respond properly to ARS. Indeed, we observed changes in several molecular key players of the hypothalamic pituitary adrenal axis, particularly influencing genomic effects mediated by the glucocorticoid receptor. This study highlights that prenatal FLX exposure influences the ability of adolescent male rats to respond to an acute challenge, thereby altering the functionality of the hypothalamic-pituitary-adrenal axis, and indicates that the prenatal manipulation may prime the response to challenging events during this critical period of life.
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Affiliation(s)
- Maria Teresa Gallo
- Department of Pharmacological and Biomolecular Sciences "Rodolfo Paoletti", Università degli Studi di Milano, Milan 20133, Italy
| | - Beatrice Dolci
- Department of Pharmacological and Biomolecular Sciences "Rodolfo Paoletti", Università degli Studi di Milano, Milan 20133, Italy
| | - Fabio Fumagalli
- Department of Pharmacological and Biomolecular Sciences "Rodolfo Paoletti", Università degli Studi di Milano, Milan 20133, Italy
| | - Paola Brivio
- Department of Pharmacological and Biomolecular Sciences "Rodolfo Paoletti", Università degli Studi di Milano, Milan 20133, Italy
| | - Francesca Calabrese
- Department of Pharmacological and Biomolecular Sciences "Rodolfo Paoletti", Università degli Studi di Milano, Milan 20133, Italy
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Hansen N, Neyazi A, Lüdecke D, Hasan A, Wiltfang J, Malchow B. Repositioning synthetic glucocorticoids in psychiatric disease associated with neural autoantibodies: a narrative review. J Neural Transm (Vienna) 2022:10.1007/s00702-022-02578-2. [PMID: 36576564 PMCID: PMC10374711 DOI: 10.1007/s00702-022-02578-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Accepted: 12/17/2022] [Indexed: 12/29/2022]
Abstract
Synthetic glucocorticoids (sGCs) are a well-investigated and standard drug therapy for disorders associated with CNS inflammation. Less is known about treating psychiatric disorders associated with neural autoantibodies. Our aim is to elucidate the repositioning of sGCs in psychiatric diseases that co-exist with neural autoantibodies. We used PubMed to identify articles for this narrative review. To our knowledge, no randomized, placebo-controlled trials have yet been conducted on applying sGC to treat neural autoantibody-associated psychiatric disorders. We describe initial results of cohort studies and single cases or case series often associated with autoantibodies against membrane-surface antigens demonstrating a largely beneficial response to sGCs either as monotherapy or polytherapy together with other immunosuppressive agents. However, sGCs may be less efficient in patients with psychiatric diseases associated with autoantibodies directed against intracellular antigens. These results reveal potential benefits of the novel usage of sGCs for the indication of neural autoantibody-associated psychiatric disease. Further large-scale randomized, placebo-controlled trials are needed to discover whether sGCs are safe, well tolerated, and beneficial in subgroups of neural autoantibody-associated psychiatric diseases.
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Affiliation(s)
- Niels Hansen
- Department of Psychiatry and Psychotherapy, University Medical Center Göttingen, Von-Siebold-Str. 5, 37075, Göttingen, Germany.
| | - Alexandra Neyazi
- Department of Psychiatry and Psychotherapy, Otto-Von-Guericke-University Magdeburg, Magdeburg, Germany
| | - Daniel Lüdecke
- Department of Psychiatry and Psychotherapy, University Hospital Hamburg Eppendorf, Hamburg, Germany
| | - Alkomiet Hasan
- Department of Psychiatry, Psychotherapy and Psychosomatics, Medical Faculty, University of Augsburg, 86156, Augsburg, Germany
| | - Jens Wiltfang
- Department of Psychiatry and Psychotherapy, University Medical Center Göttingen, Von-Siebold-Str. 5, 37075, Göttingen, Germany.,German Center for Neurodegenerative Diseases (DZNE), Von-Siebold-Str. 3a, 37075, Goettingen, Germany.,Neurosciences and Signaling Group, Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, Aveiro, Portugal
| | - Berend Malchow
- Department of Psychiatry and Psychotherapy, University Medical Center Göttingen, Von-Siebold-Str. 5, 37075, Göttingen, Germany
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Strokotova AV, Grigorieva EV. Glucocorticoid Effects on Proteoglycans and Glycosaminoglycans. Int J Mol Sci 2022; 23:ijms232415678. [PMID: 36555315 PMCID: PMC9778983 DOI: 10.3390/ijms232415678] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2022] [Revised: 11/29/2022] [Accepted: 12/08/2022] [Indexed: 12/14/2022] Open
Abstract
Glucocorticoids are steroid hormones that play diverse roles in numerous normal and pathological processes. They are actively used to treat a wide variety of diseases, including neurodegenerative and inflammatory diseases, cancers, and COVID-19, among others. However, the long-term use of glucocorticoids is associated with numerous side effects. Molecular mechanisms of these negative side effects are not completely understood. Recently, arguments have been made that one such mechanisms may be related to the influence of glucocorticoids on O-glycosylated components of the cell surface and extracellular matrix, in particular on proteoglycans and glycosaminoglycans. The potential toxic effects of glucocorticoids on these glycosylated macromolecules are particularly meaningful for brain physiology because proteoglycans/glycosaminoglycans are the main extracellular components of brain tissue. Here, we aim to review the known effects of glucocorticoids on proteoglycan expression and glycosaminoglycan content in different tissues, with a specific focus on the brain.
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Jiang C, Huang H, Kang X, Yang L, Xi Z, Sun H, Pluth MD, Yi L. NBD-based synthetic probes for sensing small molecules and proteins: design, sensing mechanisms and biological applications. Chem Soc Rev 2021; 50:7436-7495. [PMID: 34075930 PMCID: PMC8763210 DOI: 10.1039/d0cs01096k] [Citation(s) in RCA: 100] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Compounds with a nitrobenzoxadiazole (NBD) skeleton exhibit prominent useful properties including environmental sensitivity, high reactivity toward amines and biothiols (including H2S) accompanied by distinct colorimetric and fluorescent changes, fluorescence-quenching ability, and small size, all of which facilitate biomolecular sensing and self-assembly. Amines are important biological nucleophiles, and the unique activity of NBD ethers with amines has allowed for site-specific protein labelling and for the detection of enzyme activities. Both H2S and biothiols are involved in a wide range of physiological processes in mammals, and misregulation of these small molecules is associated with numerous diseases including cancers. In this review, we focus on NBD-based synthetic probes as advanced chemical tools for biomolecular sensing. Specifically, we discuss the sensing mechanisms and selectivity of the probes, the design strategies for multi-reactable multi-quenching probes, and the associated biological applications of these important constructs. We also highlight self-assembled NBD-based probes and outline future directions for NBD-based chemosensors. We hope that this comprehensive review will facilitate the development of future probes for investigating and understanding different biological processes and aid the development of potential theranostic agents.
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Affiliation(s)
- Chenyang Jiang
- State Key Laboratory of Organic-Inorganic Composites and Beijing Key Lab of Bioprocess, Beijing University of Chemical Technology (BUCT), Beijing 100029, China.
| | - Haojie Huang
- State Key Laboratory of Organic-Inorganic Composites and Beijing Key Lab of Bioprocess, Beijing University of Chemical Technology (BUCT), Beijing 100029, China.
| | - Xueying Kang
- State Key Laboratory of Organic-Inorganic Composites and Beijing Key Lab of Bioprocess, Beijing University of Chemical Technology (BUCT), Beijing 100029, China.
| | - Liu Yang
- Department of Chemistry and Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China.
| | - Zhen Xi
- State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, College of Chemistry, National Pesticide Engineering Research Center, Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China.
| | - Hongyan Sun
- Department of Chemistry and Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China. and Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong, Shenzhen 518057, China
| | - Michael D Pluth
- Department of Chemistry and Biochemistry, Materials Science Institute, Knight Campus for Accelerating Scientific Impact, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA.
| | - Long Yi
- State Key Laboratory of Organic-Inorganic Composites and Beijing Key Lab of Bioprocess, Beijing University of Chemical Technology (BUCT), Beijing 100029, China.
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Jaumotte JD, Franks AL, Bargerstock EM, Kisanga EP, Menden HL, Ghersi A, Omar M, Wang L, Rudine A, Short KL, Silswal N, Cole TJ, Sampath V, Monaghan-Nichols AP, DeFranco DB. Ciclesonide activates glucocorticoid signaling in neonatal rat lung but does not trigger adverse effects in the cortex and cerebellum. Neurobiol Dis 2021; 156:105422. [PMID: 34126164 DOI: 10.1016/j.nbd.2021.105422] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 05/28/2021] [Accepted: 06/08/2021] [Indexed: 11/15/2022] Open
Abstract
Synthetic glucocorticoids (sGCs) such as dexamethasone (DEX), while used to mitigate inflammation and disease progression in premature infants with severe bronchopulmonary dysplasia (BPD), are also associated with significant adverse neurologic effects such as reductions in myelination and abnormalities in neuroanatomical development. Ciclesonide (CIC) is a sGC prodrug approved for asthma treatment that exhibits limited systemic side effects. Carboxylesterases enriched in the lower airways convert CIC to the glucocorticoid receptor (GR) agonist des-CIC. We therefore examined whether CIC would likewise activate GR in neonatal lung but have limited adverse extra-pulmonary effects, particularly in the developing brain. Neonatal rats were administered subcutaneous injections of CIC, DEX or vehicle from postnatal days 1-5 (PND1-PND5). Systemic effects linked to DEX exposure, including reduced body and brain weight, were not observed in CIC treated neonates. Furthermore, CIC did not trigger the long-lasting reduction in myelin basic protein expression in the cerebral cortex nor cerebellar size caused by neonatal DEX exposure. Conversely, DEX and CIC were both effective at inducing the expression of select GR target genes in neonatal lung, including those implicated in lung-protective and anti-inflammatory effects. Thus, CIC is a promising, novel candidate drug to treat or prevent BPD in neonates given its activation of GR in neonatal lung and limited adverse neurodevelopmental effects. Furthermore, since sGCs such as DEX administered to pregnant women in pre-term labor can adversely affect fetal brain development, the neurological-sparing properties of CIC, make it an attractive alternative for DEX to treat pregnant women severely ill with respiratory illness, such as with asthma exacerbations or COVID-19 infections.
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Affiliation(s)
- Juliann D Jaumotte
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Pittsburgh Institute of Neurodegenerative Disease (PIND), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Alexis L Franks
- Department of Pediatrics, Division of Child Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Erin M Bargerstock
- Department of Pediatrics, Division of Newborn Medicine, UPMC Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Edwina Philip Kisanga
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Pittsburgh Institute of Neurodegenerative Disease (PIND), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Heather L Menden
- Department of Pediatrics, Division of Neonatology, Children's Mercy Kansas City, University of Missouri Kansas City School of Medicine, Kansas City, MO, USA
| | - Alexis Ghersi
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Pittsburgh Institute of Neurodegenerative Disease (PIND), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Mahmoud Omar
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Pittsburgh Institute of Neurodegenerative Disease (PIND), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Liping Wang
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Pittsburgh Institute of Neurodegenerative Disease (PIND), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Anthony Rudine
- Department of Neonatology, St. David's Medical Center, Austin, TX, USA
| | - Kelly L Short
- Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
| | - Neerupama Silswal
- Department of Biomedical Sciences, University of Missouri Kansas City School of Medicine, Kansas City, MO, USA
| | - Timothy J Cole
- Department of Biochemistry & Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
| | - Venkatesh Sampath
- Department of Pediatrics, Division of Neonatology, Children's Mercy Kansas City, University of Missouri Kansas City School of Medicine, Kansas City, MO, USA
| | - A Paula Monaghan-Nichols
- Department of Biomedical Sciences, University of Missouri Kansas City School of Medicine, Kansas City, MO, USA
| | - Donald B DeFranco
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Pittsburgh Institute of Neurodegenerative Disease (PIND), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
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8
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Rensel MA, Schlinger BA. The stressed brain: regional and stress-related corticosterone and stress-regulated gene expression in the adult zebra finch (Taeniopygia guttata). J Neuroendocrinol 2020; 32:e12852. [PMID: 32364267 PMCID: PMC7286616 DOI: 10.1111/jne.12852] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Revised: 03/16/2020] [Accepted: 04/01/2020] [Indexed: 11/30/2022]
Abstract
Glucocorticoids (CORT) are well-known as important regulators of behaviour and cognition at basal levels and under stress. However, the precise mechanisms governing CORT action and functional outcomes of this action in the brain remain unclear, particularly in model systems other than rodents. In the present study, we investigated the dynamics of CORT regulation in the zebra finch, an important model system for vocal learning, neuroplasticity and cognition. We tested the hypothesis that CORT is locally regulated in the zebra finch brain by quantifying regional and stress-related variation in total CORT across brain regions. In addition, we used an ex vivo slice culture system to test whether CORT regulates target gene expression uniquely in discrete regions of the brain. We documented a robust increase in brain CORT across regions after 30 minutes of restraint stress but, interestingly, baseline and stress-induced CORT levels varied between regions. In addition, CORT treatment of brain slice cultures differentially affected expression of three CORT target genes: it up-regulated expression of FKBP5 in most regions and SGK1 in the hypothalamus only, whereas GILZ was unaffected by CORT treatment across all brain regions investigated. The specific mechanisms producing regional variation in CORT and CORT-dependent downstream gene expression remain unknown, although these data provide additional support for the hypothesis that the songbird brain employs regulatory mechanisms that result in precise control over the influence of CORT on glucocorticoid-sensitive neural circuits.
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Affiliation(s)
- Michelle A. Rensel
- Institute for Society and Genetics, the University of California Los Angeles, Los Angeles, CA
- Laboratory of Neuroendocrinology, the University of California Los Angeles, Los Angeles, CA
- Corresponding author (MAR)
| | - Barney A. Schlinger
- Laboratory of Neuroendocrinology, the University of California Los Angeles, Los Angeles, CA
- Dept. of Integrative Biology and Physiology, the University of California Los Angeles, Los Angeles, CA
- Dept. of Ecology and Evolutionary Biology, the University of California Los Angeles, Los Angeles, CA
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9
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Thomson EM, Filiatreault A, Guénette J. Stress hormones as potential mediators of air pollutant effects on the brain: Rapid induction of glucocorticoid-responsive genes. ENVIRONMENTAL RESEARCH 2019; 178:108717. [PMID: 31520820 DOI: 10.1016/j.envres.2019.108717] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/24/2019] [Revised: 08/20/2019] [Accepted: 09/01/2019] [Indexed: 05/27/2023]
Abstract
Air pollution is associated with adverse effects on brain health including cognitive decline, dementia, anxiety, depression, and suicide. While toxicological studies have demonstrated the potential for repeated or chronic pollutant exposure to lead to disease states, characterisation of initial biological responses to exposure is needed to better understand underlying mechanisms. The brain is highly sensitive to glucocorticoids (primarily cortisol in humans, corticosterone in rodents), stress hormones that play important roles in cognition and mental health. We tested whether glucocorticoids could be implicated in central nervous system (CNS) effects of pollutant exposure by examining glucocorticoid-dependent signaling across brain regions after exposure to the common pollutant ozone. Male Fischer-344 rats were exposed for 4 h to air or 0.8 ppm ozone ± metyrapone (50 mg/kg), a drug that blocks corticosterone synthesis (n = 5/group). Key glucocorticoid-responsive genes (serum- and glucocorticoid-inducible kinase, SGK; glucocorticoid-inducible leucine zipper, GILZ), and a gene responsive to both glucocorticoids and oxidative stress (metallothionein (MT)-1), were increased by ozone in all brain regions (olfactory bulb, frontal lobe, cortex, midbrain, hippocampus, cerebellum, brainstem), correlating with plasma corticosterone levels. Metyrapone prevented the increase in SGK and GILZ, and reduced but did not eliminate the effect on MT-1, suggesting glucocorticoid-dependent and -independent regulation. Administering exogenous corticosterone (10 mg/kg) to air-exposed rats reproduced the ozone effects, confirming specificity. The results demonstrate that early pollutant effects include stress hormone-dependent signaling. As both ozone and particulate matter activate the hypothalamic-pituitary-adrenal axis, and elevated glucocorticoids are implicated in brain pathologies, stress hormones could contribute to CNS impacts of air pollutants.
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Affiliation(s)
- Errol M Thomson
- Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, K1A 0K9, Canada.
| | - Alain Filiatreault
- Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, K1A 0K9, Canada.
| | - Josée Guénette
- Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, K1A 0K9, Canada.
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Zakopoulou V, Vlaikou AM, Darsinou M, Papadopoulou Z, Theodoridou D, Papageorgiou K, Alexiou GA, Bougias H, Siafaka V, Zoccolotti P, Chroussos GP, Syrrou M, Michaelidis TM. Linking Early Life Hypothalamic-Pituitary-Adrenal Axis Functioning, Brain Asymmetries, and Personality Traits in Dyslexia: An Informative Case Study. Front Hum Neurosci 2019; 13:327. [PMID: 31632253 PMCID: PMC6779713 DOI: 10.3389/fnhum.2019.00327] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2019] [Accepted: 09/05/2019] [Indexed: 12/22/2022] Open
Abstract
Developmental dyslexia (DD) is a multi-system disorder, combining influences of susceptibility genes and environmental factors. The causative interaction between specific genetic factors, brain regions, and personality/mental disorders, as well as specific learning disabilities, has been thoroughly investigated with regard to the approach of developing a multifaceted diagnostic procedure with an intervention strategy potential. In an attempt to add new translational evidence to the interconnection of the above factors in the occurrence of DD, we performed a combinatorial analysis of brain asymmetries, personality traits, cognitive and learning skills, and expression profiles of selected genes in an adult, early diagnosed with DD, and in his son of typical development. We focused on the expression of genes, based on the assumption that the regulation of transcription may be affected by genetic and epigenetic factors. The results highlighted a potential chain link between neuroplasticity-related as well as stress-related genes, such as BDNF, Sox4, mineralocorticoid receptor (MR), and GILZ, leftward asymmetries in the amygdala and selective cerebellum lobules, and tendencies for personality disorders and dyslexia. This correlation may reflect the presence of a specific neuro-epigenetic component of DD, ensuing from the continuous, multifaceted difficulties in the acquisition of cognitive and learning skills, which in turn may act as a fostering mechanism for the onset of long-term disorders. This is in line with recent findings demonstrating a dysfunction in processes supported by rapid neural adaptation in children and adults with dyslexia. Accordingly, the co-evaluation of all the above parameters may indicate a stress-related dyslexia endophenotype that should be carefully considered for a more integrated diagnosis and effective intervention.
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Affiliation(s)
- Victoria Zakopoulou
- Department of Speech and Language Therapy, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Angeliki-Maria Vlaikou
- Laboratory of Biology, Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Marousa Darsinou
- Department of Biomedical Research, Foundation for Research and Technology-Hellas, Institute of Molecular Biology and Biotechnology, Ioannina, Greece.,Department of Biological Applications and Technologies, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Zoe Papadopoulou
- Laboratory of Biology, Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Daniela Theodoridou
- Laboratory of Biology, Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Kyriaki Papageorgiou
- Department of Biomedical Research, Foundation for Research and Technology-Hellas, Institute of Molecular Biology and Biotechnology, Ioannina, Greece.,Department of Biological Applications and Technologies, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - George A Alexiou
- Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Haralambos Bougias
- Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Vassiliki Siafaka
- Department of Speech and Language Therapy, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Pierluigi Zoccolotti
- Neuropsychology Unit, Department of Psychology, IRCCS (National Institute for Research and Treatment) Fondazione Santa Lucia, Sapienza University of Rome, Rome, Italy
| | - George P Chroussos
- First Department of Pediatrics, National and Kapodistrian University of Athens Medical School, "Aghia Sophia" Children's Hospital, Athens, Greece
| | - Maria Syrrou
- Laboratory of Biology, Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece
| | - Theologos M Michaelidis
- Department of Biomedical Research, Foundation for Research and Technology-Hellas, Institute of Molecular Biology and Biotechnology, Ioannina, Greece.,Department of Biological Applications and Technologies, School of Health Sciences, University of Ioannina, Ioannina, Greece
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11
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Bereshchenko O, Migliorati G, Bruscoli S, Riccardi C. Glucocorticoid-Induced Leucine Zipper: A Novel Anti-inflammatory Molecule. Front Pharmacol 2019; 10:308. [PMID: 30971930 PMCID: PMC6445858 DOI: 10.3389/fphar.2019.00308] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2018] [Accepted: 03/12/2019] [Indexed: 12/20/2022] Open
Abstract
Glucocorticoids (GCs) are the most commonly used drugs for treatment of autoimmune and inflammatory diseases. Their efficacy is due to their ability to bind cytoplasmic receptors (glucocorticoid receptors, GR) and other cytoplasmic proteins, thus regulating gene expression. Although GCs are potent life-saving drugs, their therapeutic effects are transitory and chronic use of GCs is accompanied by serious side effects. Therefore, new drugs are needed to replace GCs. We have identified a gene, glucocorticoid-induced leucine zipper (GILZ or tsc22d3), that is rapidly and invariably induced by GCs. Human GILZ is a 135-amino acid protein that mediates many GC effects, including inhibition of the NF-κB and MAPK pathways. Similar to GCs, GILZ exerts anti-inflammatory activity in experimental disease models, including inflammatory bowel diseases and arthritis. While transgenic mice that overexpress GILZ are more resistant, GILZ knockout mice develop worse inflammatory diseases. Moreover, the anti-inflammatory effect of GCs is attenuated in GILZ-deficient mice. Importantly, in vivo delivery of recombinant GILZ protein cured colitis and facilitated resolution of lipopolysaccharide-induced inflammation without apparent toxic effects. A synthetic GILZ-derived peptide, corresponding to the GILZ region that interacts with NF-κB, was able to suppress experimental autoimmune encephalomyelitis. Collectively, these findings indicate that GILZ is an anti-inflammatory molecule that may serve as the basis for designing new therapeutic approaches to inflammatory diseases.
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Affiliation(s)
- Oxana Bereshchenko
- Department of Surgery and Biomedical Sciences, University of Perugia, Perugia, Italy
| | - Graziella Migliorati
- Section of Pharmacology, Department of Medicine, University of Perugia, Perugia, Italy
| | - Stefano Bruscoli
- Section of Pharmacology, Department of Medicine, University of Perugia, Perugia, Italy
| | - Carlo Riccardi
- Section of Pharmacology, Department of Medicine, University of Perugia, Perugia, Italy
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12
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Peripheral blood GILZ mRNA levels in depression and following electroconvulsive therapy. Psychoneuroendocrinology 2019; 101:304-310. [PMID: 30602137 DOI: 10.1016/j.psyneuen.2018.12.234] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/23/2018] [Revised: 12/20/2018] [Accepted: 12/22/2018] [Indexed: 12/12/2022]
Abstract
Dysregulation of the hypothalamic-pituitary-adrenocortical (HPA)-axis is commonly observed in patients with depression. The delayed feedback system that mediates inhibition of HPA-axis activation is regulated by glucocorticoid receptors (GRs) found in stress-responsive areas of the brain. Glucocorticoid-induced leucine zipper (GILZ) is a key molecule in glucocorticoid biology and is thought to mediate the downstream anti-inflammatory effects of GRs. Previous reports suggest that GILZ levels are altered in the blood and brains of patients with, and animal models of, depression. However, no study has yet investigated the effects of antidepressant treatment on GILZ. Therefore, our aim was to examine peripheral blood GILZ mRNA levels in patients with depression (n = 88) compared to age- and sex-matched healthy controls (n = 63), and in patients with depression following treatment with a course of electroconvulsive therapy (ECT). We also assessed the relationship between GILZ and mood and clinical outcomes following ECT. GILZ mRNA levels were assessed using qRT-PCR. GILZ levels were found to be significantly lower in patients with depression compared to controls (p < 0.002), and ECT further decreased GILZ levels (p = 0.05). Both of these results survived adjustment for potential covariates. However, we found no association between GILZ and mood scores. Overall, these results suggest that GILZ is involved in the pathophysiology of depression and the peripheral molecular response to ECT.
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Witek E, Hickman D, Lahiri DK, Srinivasan M. Glucocorticoid Induced Leucine Zipper in Lipopolysaccharide Induced Neuroinflammation. Front Aging Neurosci 2019; 10:432. [PMID: 30740047 PMCID: PMC6355683 DOI: 10.3389/fnagi.2018.00432] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2018] [Accepted: 12/17/2018] [Indexed: 11/13/2022] Open
Abstract
Glucocorticoids (GCs) are steroid hormones secreted as the end-product of the neuroendocrine stress cascade. Both absence and elevated GC mediate neurotoxic responses, suggesting that a narrow window ranging from physiological to slightly high GC mediate protective responses. The beneficial effects of GC are attributed to the transactivation of regulatory proteins and inhibition mediated by glucocorticoid receptor (GR) interactions with other co-factors. The glucocorticoid induced leucine zipper (GILZ) is a gene strongly upregulated by GC and mediates many of the anti-inflammatory and anti-proliferative effects of GC. Although GILZ is constitutively expressed in many tissues including the brain, the expression has been shown to occur with varying dynamics suggesting that the local milieu modulates its expression with consequent effects on cellular responses. Here we investigated the expression profile of GILZ in lipopolysaccharide (LPS) mediated neuroinflammation model of Alzheimer's disease (AD). Our data suggest that the GILZ expression is downregulated in neuroinflammation correlating inversely with the pro-inflammatory cytokines and innate immune responses.
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Affiliation(s)
- Emily Witek
- Laboratory Animal Resources and Center, Department of Internal Medicine, Indiana University School of Medicine, Indianapolis, IN, United States
| | - Debra Hickman
- Laboratory Animal Resources and Center, Department of Internal Medicine, Indiana University School of Medicine, Indianapolis, IN, United States
| | - Debomoy K Lahiri
- Department of Psychiatry, Stark Neuroscience Research Institute, Indiana University School of Medicine, Indianapolis, IN, United States
| | - Mythily Srinivasan
- Department of Oral Pathology, Medicine and Radiology, Indiana University School of Dentistry and Indiana University-Purdue University Indianapolis, Indiana University School of Medicine, Indianapolis, IN, United States
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Baban B, Marchetti C, Khodadadi H, Malik A, Emami G, Lin PC, Arbab AS, Riccardi C, Mozaffari MS. Glucocorticoid-Induced Leucine Zipper Promotes Neutrophil and T-Cell Polarization with Protective Effects in Acute Kidney Injury. J Pharmacol Exp Ther 2018; 367:483-493. [PMID: 30301736 DOI: 10.1124/jpet.118.251371] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2018] [Accepted: 10/01/2018] [Indexed: 12/11/2022] Open
Abstract
The glucocorticoid-induced leucine zipper (GILZ) mediates anti-inflammatory effects of glucocorticoids. Acute kidney injury (AKI) mobilizes immune/inflammatory mechanisms, causing tissue injury, but the impact of GILZ in AKI is not known. Neutrophils play context-specific proinflammatory [type 1 neutrophil (N1)] and anti-inflammatory [type 2 neutrophil (N2)] functional roles. Also, regulatory T lymphocytes (Tregs) and regulatory T-17 (Treg17) cells exert counterinflammatory effects, including the suppression of effector T lymphocytes [e.g., T-helper (Th) 17 cells]. Thus, utilizing cell preparations of mice kidneys subjected to AKI or sham operation, we determined the effects of GILZ on T cells and neutrophil subtypes in the context of its renoprotective effect; these studies used the transactivator of transcription (TAT)-GILZ or the TAT peptide. AKI increased N1 and Th-17 cells but reduced N2, Tregs, and Treg17 cells in association with increased interleukin (IL)-17+ but reduced IL-10+ cells accompanied with the disruption of mitochondrial membrane potential (ψ m) and increased apoptosis/necrosis compared with sham kidneys. TAT-GILZ, compared with TAT, treatment reduced N1 and Th-17 cells but increased N2 and Tregs, without affecting Treg17 cells, in association with a reduction in IL-17+ cells but an increase in IL-10+ cells; TAT-GILZ caused less disruption of ψ m and reduced cell death in AKI. Importantly, TAT-GILZ increased perfusion of the ischemic-reperfused kidney but reduced tissue edema compared with TAT. Utilizing splenic T cells and bone marrow-derived neutrophils, we further showed marked reduction in the proliferation of Th cells in response to TAT-GILZ compared with response to TAT. Collectively, the results indicate that GILZ exerts renoprotection accompanied by the upregulation of the regulatory/suppressive arm of immunity in AKI, likely via regulating cross talk between T cells and neutrophils.
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Affiliation(s)
- Babak Baban
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
| | - Cristina Marchetti
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
| | - Hesam Khodadadi
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
| | - Aneeq Malik
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
| | - Golnaz Emami
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
| | - Ping-Chang Lin
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
| | - Ali S Arbab
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
| | - Carlo Riccardi
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
| | - Mahmood S Mozaffari
- Department of Oral Biology and Diagnostic Sciences, Dental College of Georgia (B.B., H.K., A.M., G.E., M.S.M.) and Georgia Cancer Center (P.-C.L., A.S.A.), Augusta University, Augusta, Georgia; and Department of Medicine, University of Perugia, Perugia, Italy (C.M., C.R.)
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15
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Fukushima R, Yamamoto J, Ishikawa H, Kinjo M. Two-detector number and brightness analysis reveals spatio-temporal oligomerization of proteins in living cells. Methods 2018; 140-141:161-171. [PMID: 29572069 DOI: 10.1016/j.ymeth.2018.03.007] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2017] [Revised: 03/06/2018] [Accepted: 03/16/2018] [Indexed: 01/10/2023] Open
Abstract
Number and brightness analysis (N&B) is a useful tool for the simultaneous visualization of protein oligomers and their localization, with single-molecule sensitivity. N&B determines particle brightness (fluorescence intensity per particle) and maps the spatial distribution of fluorescently labeled proteins by performing statistical analyses of the image series obtained using laser scanning microscopy. The brightness map reveals presence of the oligomers of the targeted protein and their distribution in living cells. However, even when corrections are applied, conventional N&B is affected by afterpulsing, shot noise, thermal noise, dead time, and overestimation of particle brightness when the concentration of the fluorescent particles changes during measurement. The drawbacks of conventional N&B can be circumvented by using two detectors, a novel approach that we henceforth call two-detector number and brightness analysis (TD-N&B), and introducing a linear regression of fluorescence intensity. This statistically eliminates the effect of noise from the detectors, and ensures that the correct particle brightness is obtained. Our method was theoretically assessed by numerical simulations and experimentally validated using a dilution series of purified enhanced green fluorescent protein (EGFP), EGFP tandem oligomers in cell lysate, and EGFP tandem oligomers in living cells. Furthermore, this method was used to characterize the complex process of ligand-induced glucocorticoid receptor dimerization and their translocation to the cell nucleus in live cells. Our method can be applied to other oligomer-forming proteins in cell signaling, or to aggregations of proteins such as those that cause neurodegenerative diseases.
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Affiliation(s)
- Ryosuke Fukushima
- Laboratory of Molecular Cell Dynamics, Graduate School of Life Science, Hokkaido University, Sapporo 001-0021, Japan
| | - Johtaro Yamamoto
- Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan
| | - Hideto Ishikawa
- Laboratory of Molecular Cell Dynamics, Graduate School of Life Science, Hokkaido University, Sapporo 001-0021, Japan
| | - Masataka Kinjo
- Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
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